ES2650917T3 - Biomarker proteins of atypical hemolytic uremic syndrome (SUHA) - Google Patents

Abstract

A complement inhibitor, which is an anti-C5 antibody or a C5-binding fragment thereof, for use in a method of treating atypical hemolytic uremic syndrome (aHUS), the method comprising: (a) determining the concentration of at least two biomarker proteins associated with aHUS in a biological fluid obtained from a subject who has, is suspected of having or is at risk of developing aHUS, in which the biomarker proteins associated with aHUS are TNFR1 and at least one other biomarker protein associated with aHUS selected de: CXCL10, MCP-1, IFN-γ, IL-6, a proteolytic fragment of complement component factor B, soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, Factor von Willebrand (vWF), complement component C5a, ß2 (ß2M) microglobulin, clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9, KIM- 1, soluble CD40 ligand (sCD40L), ICAM-1, IL-1 beta, IL-12 p70, IL-8 and vascular endothelial cell growth factor (VEGF); and (b) administering the complement inhibitor to the subject in an amount and frequency sufficient to reduce the concentration of the at least two biomarker proteins associated with aHUS, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before complement inhibitor treatment.

Description

DESCRIPTION

Biomarker proteins of atypical hemolytic uremic syndrome (SUHA)

Technical Field 5

The field of the invention is medicine, immunology, molecular biology and protein chemistry.

Background

 10

Hemolytic uremic syndrome (HUS) is characterized by thrombocytopenia, microangiopathic hemolytic anemia and acute renal failure. SUH is classified as one of two types: SUH associated with diarrhea (SUH D +; also called SUH of E. coli producing Shiga toxin (STEC) or typical SUH) and non-diarrheal or atypical SUH (SUHa). SUH D + is the most common form, which represents more than 90% of cases and is caused by a previous disease with a Shiga-type toxin-producing bacterium, for example, E. coli O157: H7. SUHa is unusual 15 and has a mortality rate of up to 25%. Many patients with this disease will suffer permanent renal or neurological insufficiency, for example, at least 50% of patients with SUHa progress to end-stage renal failure (SRF). See, for example, Kavanagh et al. (2006) British Medical Bulletin 77 and 78: 5-22.

 twenty

SUHa can be genetic, acquired or idiopathic. Inheritable forms of SUHa may be associated with mutations in various components of the human complement including, for example, complement factor H (CFH), membrane cofactor protein (MCP), complement factor I (CFI), protein binding to C4b (C4BP), complement factor B (CFB) and complement component 3 (C3). See, for example, Caprioli et al. (2006) Blood 108: 1267-1279. Certain mutations in the gene encoding CD55, although not yet involved in SUHa, 25 are associated with the severity of SUHa. See, for example, Esparza-Gordillo et al. (2005) Hum Mol Genet 14: 703-712.

Until recently, treatment options for patients with SUHa were limited and often involved plasma infusion or plasma exchange. In some cases, patients with SUHa undergo uni or bilateral nephrectomy or renal transplantation (see Artz et al. (2003) Transplantation 76: 821-826). However, the recurrence of the disease in treated patients is common. Recently, the treatment of patients with SUHa with the drug Soliris® was approved in the United States of America and in Europe. Despite finally having a useful drug for the treatment of patients with SUHa, there is still a need to diagnose patients with SUHa, as well as monitor the progression and decrease of SUHa.

 35

Okamoto et al. (2011) Journal of Immunoassay and Immunochemistry 32 (2): 145-155 describes an ELISA assay used to show that the levels of sTNFRI and sTNFRII in urine of patients with HUS were markedly higher than those of healthy children since the onset of uremic syndrome Hemolytic diarrhea positive (SUH D +).

Summary 40

The present disclosure provides, among other things, a variety of proteins whose activity and / or concentration in a biological liquid is abnormal in patients suffering from SUHa and / or patients with SUHa receiving complement inhibitor therapy. Hereinafter these proteins are referred to as "biomarker proteins associated with SUHa" or "biomarker proteins of SUHa". For example, the 45 inventors have observed that the concentrations and / or activities of various proteins in the blood (for example, serum and / or plasma) and urine are abnormal in patients with SUHa. The inventors have also observed that, after administration of an anti-C5 antagonist antibody (eculizumab) to a human being, the concentrations of a subset of these proteins change. In some cases, the concentration of one or more of the proteins is normalized. Although the disclosure is not linked to any particular theory or mechanism of action, the inventors believe that the supervision of a patient treated with a complement inhibitor (such as an anti-C5 antibody) with respect to a change in concentration of one or more of these proteins, biomarker proteins of SUHa, is useful for, for example, diagnosing that a patient has or is at risk of developing SUHa. Monitoring the status of one or more of these biomarker proteins may also be useful in determining whether a patient with SUHa is responding to therapy with a complement inhibitor. In addition, the evaluation of the status of one or more of the biomarkers is also useful for identifying a dose, a threshold dose, of a complement inhibitor, such as an anti-C5 antibody, which by virtue of its effect on the concentration of a or more of the biomarker proteins of SUHa in humans is sufficient to achieve a clinically significant effect on the disease (i.e., sufficient to treat a disease associated with complement, such as SUHa). 60

Accordingly, in one aspect, the present invention provides a complement inhibitor, which is an anti-C5 antibody or a C5 binding fragment thereof, for use in a method of treating atypical hemolytic uremic syndrome (SUHa), the method comprising :

 65

(a) determining the concentration of at least two biomarker proteins associated with SUHa in a biological liquid obtained from a subject that has, is suspected of having or is at risk of developing, SUHa, in which the biomarker proteins associated with SUHa are TNFR1 and at least one other biomarker protein associated with SUHa selected from: CXCL10, MCP-1, IFNγ, IL-6, a proteolytic fragment of complement component component B, soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer , thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, microglobulin β2 5 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP- 1), albumin, CXCL9, KIM-1, soluble CD40 ligand (sCD40L), ICAM-1, IL-1 beta, IL-12 p70, IL-8 and vascular endothelial cell growth factor (VEGF); Y

(b) administer to the subject the complement inhibitor in an amount and with a frequency sufficient to reduce the concentration of the at least two biomarker proteins associated with SUHa, compared with 10 the concentration in a sample of the same type of biological liquid obtained from the subject before treatment with complement inhibitor.

In one embodiment, the reduced concentration of the at least two biomarker proteins associated with SUHa can occur within two weeks or two months after the first administration of the complement inhibitor.

The subject may have received dialysis at least once in the three months immediately prior to treatment with the complement inhibitor, or experience the first manifestation of acute SUHa.

 twenty

In another aspect, the invention provides a method for monitoring the sensitivity of a subject to treatment with a complement inhibitor, which is an anti-C5 antibody or a C5 binding fragment thereof, the method comprising: determining the concentration of at least two biomarker proteins associated with SUHa in a biological liquid obtained from the subject, in which:

 25

(a) the biomarker proteins associated with SUHa are TNFR1 and at least one other biomarker protein associated with SUHa selected from: CXCL10, MCP-1, IFN-γ, IL-6, a proteolytic fragment of the component component of complement B, soluble C5b9 ( sC5b9), prothrombin fragment F1 + 2, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9 and KIM-1;

(b) the subject has, is suspected of having, or is at risk of developing SUHa;

(c) the subject has been treated or is being treated with the complement inhibitor; Y

(d) a reduced concentration of the at least two biomarker proteins associated with SUHa compared to the concentration in a sample of biological liquid of the same type obtained from the subject prior to treatment with the complement inhibitor indicates that the subject is sensitive to treatment. With the inhibitor.

In the above methods, the subject may be a subject that is treated chronically with a complement inhibitor.

 40

In the above methods, the complement inhibitor can be an antibody or an antigen-binding fragment of an antibody that:

(i) is selected from a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a completely human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab 'fragment and an F (ab') 2 fragment;

(ii) inhibits the cleavage of C5 in fragments C5a and C5b; or

(iii) is the eculizumab antibody or the pexelizumab antigen binding fragment.

 fifty

In a further aspect, the invention provides a method for diagnosing that a subject has or is at risk of developing atypical hemolytic uremic syndrome (SUHa), the method comprising: determining the concentration of at least two biomarker proteins associated with SUHa in a liquid Biological obtained from a subject, in which the biomarker proteins associated with SUHa are TNFR1 and at least one other biomarker protein associated with SUHa selected from: a proteolytic fragment of component component of complement B, soluble C5b9 (sC5b9), thrombomodulin, VCAM- 1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, IFN-γ, ICAM-1, IL-1 beta, IL- 12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF) , IL-6, albumin, IL-8 and CCL5 in which a high concentration of the at least two biomarker proteins associated with SUHa compared to the concentration of biomarker proteins associated with SUHa in a biological liquid control sample of the The same type indicates that the subject has, or is at risk of developing, SUHa.

In the above methods, the at least one other biomarker protein associated with additional SUHa can be selected from: a soluble factor B, C5a, C5b-9 proteolytic fragment (sC5b-9), VCAM-1, thrombomodulin, prothrombin fragments 1 and 2 (F1 + 2), D-dimer, clusterin, TIMP-1, FABP-1, microglobulin beta-2 (b2m) and cystatin C, in which VCAM-1 is soluble VCAM-1 (sVCAM-1).

In the above methods, the at least two biomarker proteins associated with SUHa can be measured using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). 5

In the above methods, the biological liquid can be blood, a blood fraction, such as serum or plasma, or urine. The blood fraction is preferably serum.

In the above methods, the concentration of at least one biomarker associated with SUHa can be measured in one 10 or more types of biological fluid; or the concentration of a first of the at least two biomarker proteins associated with SUHa can be measured in one type of biological liquid and the concentration of a second of the at least two biomarker proteins of SUHa can be measured in a second type of liquid.

In the above methods, it is preferred that:

(i) determine the concentration of CXCL9, CXCL10, IFN-γ, MCP-1, CCL5, sCD40L or TNFR1 in the subject's serum, in which TNFR1 is soluble TNFR1 (sTNFR1);

(ii) determine the concentration of at least one of β2 (β2M) microglobulin, clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin and KIM-1 in the subject's urine 20;

(iii) determine the concentration of NGAL, a proteolytic fragment of the complement component component B, soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, D-dimer, thrombomodulin or von Willebrand factor (vWF) in the plasma of the subject; I

(iv) determine the concentration of Ba in plasma obtained from the subject. 25

In the above methods, the normal control concentration of sTNFR1 is less than 2000 pg / ml. The concentration of sTNFR1 in the biological sample is considered high when it is: (i) at least twice as high as the control concentration of sTNFR1; or (ii) at least 10,000 pg / ml.

 30

In a further aspect, the invention provides the use of a diagnostic kit in the diagnosis of SUHa, in which the diagnostic kit comprises: (a) a test plate and (b) at least three binding agents, in which each binding agent binds with a different biological analyte protein, in which the analyte proteins are selected from: a proteolytic fragment of the complement component component B, soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, factor von Willebrand (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL- 12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, Vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5, and one of the proteins is TNFR-1.

 40

It is preferred that the binding agent be an antibody or an antigen binding fragment thereof.

The disclosure presents a method for monitoring or evaluating the status of biomarker proteins associated with atypical hemolytic uremic syndrome (SUHa) in a subject (for example, a mammal such as a human being) or a method for assessing one or both of the concentration and the level of activity of at least one biomarker protein associated with atypical hemolytic uremic syndrome (SUHa) in a subject. The method comprises measuring in a biological liquid obtained from the subject one or both of (i) the concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) biomarker protein associated with SUHa in the biological liquid, in which the biomarker proteins associated with SUHa are any of the biomarkers set forth in Table 1, for example , one selected from the group consisting of: a proteolytic fragment of the complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), ligand of Soluble CD40 (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, cell growth factor Vascular endothelial (VEGF), 55 IL-6, albumin, IL-8 and CCL5. The subject may be, for example, a human being who has, is suspected of having or is at risk of developing, SUHa. The subject may be one that has been treated (or is being treated) with a complement inhibitor (eg, an inhibitor of complement component C5 such as an anti C5 antibody). The treatment may have been less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days) before obtaining the sample of the subject. 60 The method may also include the step of determining if the subject has or is at risk of developing SUHa. When the subject has been treated or is being treated with a complement inhibitor (for example, an anti-C5 antibody) in a predetermined dosage schedule, the method may also include determining whether the patient is (therapeutically) sensitive to the inhibitory therapy of the complement.

 65

The disclosure presents a method for monitoring or evaluating the status of biomarker proteins associated with atypical hemolytic uremic syndrome (SUHa) in a subject (for example, a mammal such as a human being) or a method for assessing one or both of the concentration and the level of activity of at least one biomarker protein associated with atypical hemolytic uremic syndrome (SUHa) in a subject. The method comprises: (A) measuring in a biological liquid obtained from the subject the concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20) biomarker proteins associated with SUHa in the biological liquid, in which the biomarker proteins associated with SUHa are any of the biomarkers set forth in Table 1, for example, one selected from the group consisting of: a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand ( sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, component of complement C5a, microglobulin β2 (β2M ), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, endothelial cell growth factor l vascular (VEGF), IL-6, albumin, IL-8 and CCL5; and (B) record (for example, in an electronic patient register) the results of the measurement or measurements or communicate the results of the measurement or measurements to the subject, the subject's guardian or a medical professional whose position has been put on the subject. The subject may be, for example, a human being who has, is suspected of having, or is at risk of developing, SUHa. The subject may be one that has been treated (or is being treated) with a complement inhibitor (eg, an inhibitor of complement component C5 such as an anti C5 antibody). The treatment may have occurred less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days) before obtaining the sample of the subject. The method may also include the step of determining if the subject has or is at risk of developing 20 SUHa. When the subject has been treated or is being treated with a complement inhibitor (for example, an anti-C5 antibody) in a predetermined dosage schedule, the method may also include determining whether the patient is (therapeutically) sensitive to the inhibitory therapy of the complement.

The disclosure presents a method to monitor or determine if a patient is at risk of developing thrombotic microangiopathy. The method includes (A) measuring in a biological liquid obtained from the subject the concentration of at least one (for example, at least two, three, four) biomarker protein associated with thrombosis or coagulation in the biological liquid, in which the biomarker proteins they are any of the biomarkers set forth in Table 1 or Table 11, for example, F1 + 2 or D-dimer; and (B) record (for example, in an electronic patient register) the results of the measurement or measurements or communicate the results of the measurement or the 30 measurements to the subject, the subject's guardian or a medical professional in whose charge He has put the subject. The subject may be, for example, a human being who has, is suspected of having, or is at risk of developing SUHa. The subject may be one that has been treated (or is being treated) with a complement inhibitor (eg, an inhibitor of complement component C5 such as an anti C5 antibody). The treatment may have occurred less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 35 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days) before obtaining the sample of the subject. The method may also include the step of determining if the subject has or is at risk of developing SUHa (or confirming a diagnosis of SUHa) using any of the methods described herein. When the subject has been treated or is being treated with a complement inhibitor (for example, an anti-C5 antibody) in a predetermined dosage schedule, the method may also include determining whether the patient is (therapeutically) sensitive to inhibitory therapy. of complement, that is, there is a reduction in the concentration of one or more of the biomarkers associated with thrombosis or coagulation after treatment with the complement inhibitor.

The disclosure presents a method for monitoring or evaluating the status of biomarker proteins associated with atypical hemolytic uremic syndrome (SUHa) in a subject (for example, a mammal such as a human being) or a method for assessing one or both of the concentration and the level of activity of at least one biomarker protein associated with atypical hemolytic uremic syndrome (SUHa) in a subject. The method comprises: (A) measuring in a biological liquid obtained from the subject the concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12 or 13 ) biomarker protein associated with SUHa in the biological liquid, in which the biomarker proteins associated with SUHa are any of the biomarkers 50 shown in Table 1, for example, one selected from the group consisting of: a proteolytic fragment of the component factor of the complement B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP -1, and fatty acid binding protein 1 (FABP-1); and (B) record (for example, in an electronic patient register) the results of the measurement or measurements or communicate the results of the measurement or measurements to the subject, the subject's guardian or a medical professional in whose charge He has put the subject. The subject may be, for example, a human being who has, is suspected of having or is at risk of developing, SUHa. The subject may be one that has been treated (or is being treated) with a complement inhibitor (eg, an inhibitor of complement component C5 such as an anti C5 antibody). The treatment may have been less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 60 3, 2 or 1 days) before obtaining the sample of the subject. The method may also include the step of determining if the subject has or is at risk of developing SUHa. When the subject has been treated or is being treated with a complement inhibitor (for example, an anti-C5 antibody) in a predetermined dosage schedule, the method may also include determining whether the patient is (therapeutically) sensitive to the inhibitory therapy of the complement. 65

Any of the methods described herein may further comprise determining whether the subject has or is at risk of developing SUHa. In some embodiments, a high concentration, compared to the concentration in a normal control biological liquid of the same type, of at least one of Ba, sC5b-9, C5a, sCD40L, prothrombin fragment F1 + 2, D-dimer, thrombomodulin, VCAM-1, vWF, FABP-1, p2M, clusterin, cystatin C, TIMP-1, albumin, NGAL, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF, CCL5, IL - 6, I FNγ, 5 indicates that the subject has, or is at risk of developing, SUHa.

Any of the methods described herein includes determining whether the subject has responded to treatment with the complement inhibitor. In some embodiments, (a) a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN -γ, a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF ), complement component C5a, sC5b9, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1 ; or (b) an increased concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of CCL5, indicates that the subject is sensitive to the treatment with the inhibitor.

The disclosure presents a method for monitoring the sensitivity of a subject (for example, a mammal such as a human being) to treatment with a complement component C5 inhibitor. The method includes: measuring the concentration of at least two biomarker proteins associated with SUHa in a biological liquid, in which the biomarker proteins associated with SUHa are any of those set forth in Table 1, for example, one selected from the group consisting of in: a proteolytic fragment of complement component component B (eg, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), fragment of prothrombin F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 25 beta, IL-12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL -8 and CCL5. The biological fluid is obtained from a subject: (i) who has, is suspected of having or is at risk of developing SUHa and (ii) that is being treated (or that has been treated, for example, recently) with an inhibitor of complement component C5 in a predetermined dosage schedule 30. According to said methods, (a) a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor ( vWF), complement component C5a, microglobulin β2 35 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1 ; or (b) an increased concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject prior to treatment with the inhibitor, of CCL5, indicates that the subject is sensitive to treatment with the inhibitor.

 40

Any of the methods described herein includes determining whether the subject has responded to treatment with the complement inhibitor. In some embodiments, a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject prior to treatment with the inhibitor, of at least one of a proteolytic fragment of the complement factor B component (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-45 dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP-1 and protein fatty acid binding 1 (FABP-1).

The disclosure presents a method for monitoring the sensitivity of a subject to treatment with a complement inhibitor, in which the method comprises: determining the concentration of at least two biomarker proteins associated with SUHa in a biological liquid obtained from the subject, in the that biomarker proteins associated with SUHa are selected from the group consisting of: CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, a proteolytic fragment of complement component component B (eg, Ba or Bb), C5b9 soluble (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP- 1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9, KIM-1 and CCL5. The subject has, is suspected of having, or is at risk of developing SUHa and the subject has been treated or is being treated with a complement inhibitor. (A) a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, IL- 6, a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 60 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF ), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9 and KIM-1; or (B) an increased concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of CCL5, indicates that the subject is sensitive to treatment with the inhibitor. 65

The disclosure presents a method to reduce the number, frequency or occurrence, probability of onset or risk of development of TMA, using a complement inhibitor in a manner sufficient to induce a physiological change in at least two biomarker proteins associated with thrombosis or coagulation. The method includes: (a) determining the concentration of at least two biomarker proteins in a biological liquid obtained from the subject, in which the biomarker proteins are selected from Table 1 or 11 and are related to thrombosis and / or coagulation. (for example, D-dimer or F1 + 2); and (b) administer to a subject who has, suspected of having or being at risk of developing TMA a complement inhibitor in an amount and with sufficient frequency to cause a physiological change in at least each of two (2) of biomarker proteins, in which the physiological change is a reduction in the concentration of at least two biomarker proteins in relation to the concentration of the markers in an equivalent biological sample obtained from the subject before treatment with the complement inhibitor. The method may include measuring the concentration of biomarkers both before and after treatment.

The disclosure presents a method to determine if a patient with SUHa treated with a complement inhibitor in a predetermined dosage schedule needs: (i) treatment with a different complement inhibitor or (ii) treatment with the same complement inhibitor in a different dosing schedule. The method comprises: (A) determining whether the patient with SUHa is sensitive to treatment with the complement inhibitor in the predetermined dosage schedule, in which the determination comprises: measuring in the biological liquid obtained from the subject one or both of the concentration and the activity of at least two biomarker proteins associated with SUHa in the biological liquid, in which the biomarker proteins associated with 20 SUHa are selected from the group consisting of: a proteolytic fragment of the complement factor component B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 ( FABP-1), CXCL9, KIM-1, IL-18, cel growth factor vascular endothelial ular 25 (VEGF), IL-6, albumin, IL-8 and CCL5, and in which: (a) a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before of treatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment of the complement component component B (e.g., Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, sC5b9, 30 microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, protein of fatty acid binding 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1; or (b) an increased concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject prior to treatment with the inhibitor, of CCL5, indicates that the subject is sensitive to treatment with the inhibitor; and (B) if the patient is not sensitive to treatment with the complement inhibitor, administer to the patient a different complement inhibitor or the same complement inhibitor at a higher dose or in a more frequent dosing schedule compared to the program. Default dosage.

The disclosure presents a method to determine if a patient with SUHa treated with a complement inhibitor in a predetermined dosage schedule needs: (i) treatment with a different complement inhibitor or (ii) treatment with the same complement inhibitor in a different dosing schedule. The method comprises: (A) determining whether the patient with SUHa is sensitive to treatment with the complement inhibitor in the predetermined dosage schedule, in which the determination comprises: measuring in the biological liquid obtained from the subject one or both of the concentration and the activity of at least two biomarker proteins associated with SUHa in the biological liquid, in which the biomarker proteins associated with 45 SUHa are selected from the group consisting of: a proteolytic fragment of the complement factor component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1) and in which: (a) a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of at minus one of a 50 proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1), indicates that the subject is sensitive to treatment with the inhibitor; and (B) if the patient is not sensitive to treatment with the complement inhibitor, administer to the patient a different complement inhibitor or the same complement inhibitor at a higher dose or in a more frequent dosing schedule compared to the program. Default dosage.

The concentration of one or more of the proteins can be measured using, for example, an immunoassay (for example, enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), Western blot or point transfer) or matrix of cytometric beads ( CBA, see work examples). Said 60 methods as well as kits useful for performing the methods are described herein. Suitable methods for measuring vWF activity are described in the art and are described herein.

In some embodiments of any of the methods described herein, the concentrations of at least five biomarker proteins associated with individual SUHa are measured. In some embodiments of any of the methods described herein, the concentrations of at least ten biomarker proteins associated with individual SUHa are measured. In some embodiments of any of the methods described herein, the concentrations of at least 15 biomarker proteins associated with individual SUHa are measured. In some embodiments of any of the methods described herein, the concentrations of at least 20 biomarker proteins associated with individual SUHa are measured. 5

In some embodiments of any of the methods described herein, the biological liquid is blood. In some embodiments, the biological liquid is a fraction of blood, for example, serum or plasma. In some embodiments, the biological fluid is urine. In some embodiments of any of the methods described herein, all measurements are made in a biological liquid. In some embodiments of any of the methods described herein, measurements are made in at least two different biological liquids obtained from the subject. In some embodiments, the concentrations of at least two biomarker proteins associated with individual SUHa are measured and the concentration of the first biomarker protein associated with SUHa in one type of biological liquid is measured and the second biomarker protein associated with SUHa is measured in a second type of biological fluid. fifteen

In some embodiments of any of the methods described herein, the concentrations of at least two (for example, at least three, four or all) of IFN-γ, ICAM-1, IL-1 beta and IL- are measured 12 p70. In some embodiments of any of the methods described herein, the concentrations of both Ba and sC5b9 are measured. In some embodiments of any of the methods described herein, the concentrations of one or both of C5a and C5b9 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two (eg, at least three, four, five, six or all) of β2M, clusterin, cystatin C, NAG, TIMP- are measured. 1, NGAL and FABP-1. In some embodiments of any of the methods described herein, the concentrations of CXCL10, CXCL9 and / or KIM-1 are measured. In some embodiments of any of the methods described herein, the concentrations of one or both of D-dimer and F1 + 2 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two (for example, at least three, four or all) of sCD40L, prothrombin fragment F1 + 2 and D-dimer are measured. In some embodiments of any of the methods described herein, the concentrations of thrombomodulin, VCAM-1 and / or vWF are measured. In some embodiments of any of the methods described herein, the concentrations of CXCL10, MCP-1 and / or TNFR1 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two (for example, at least three, four or all) of IFN-γ, ICAM-1, IL-1 beta and IL- are measured 12 p70.

In some embodiments of any of the methods described herein, the concentrations of one or more of CXCL9, CXCL10, IL-1 beta, IL-12 p70, IFN-γ, MCP-1, CCL5, sCD40L and / or sTNFR1 in the subject's serum. In some embodiments, the concentrations of one or more of the complement component C5a, sC5b9, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1) are measured ), CXCL10, CXCL9 and / or KIM-1 in the subject's urine. In some embodiments of any of the methods described herein, the concentrations of one or more of NGAL, a proteolytic fragment of complement component component B (e.g., Ba or Bb), soluble C5b9 (sC5b9), are measured, Prothrombin fragment F1 + 2, D-dimer, thrombomodulin and / or von Willebrand factor (vWF) in the subject's plasma.

In some embodiments, the concentrations of two or more (for example, three, four, five, six, seven, 45, eight, nine, 10, 11, 12 or 13) of a proteolytic fragment of the complement component factor B ( for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP-1 and 1 fatty acid binding protein (FABP-1).

In some embodiments of any of the methods described herein, the concentration of at least two of the group consisting of Ba, sC5b-9 and C5a is measured. In some embodiments of any of the methods described herein, the concentration of one or both of Ba and sC5b9 is measured. In some embodiments of any of the methods described herein, the concentration of one or both of C5a and C5b9 is measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of β2M, clusterin, cystatin C, albumin, TIMP-1, NGAL and FABP-1 are measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, IL-6 and IFNγ are measured . In some embodiments of any of the methods described herein, the concentration of one or both of d-dimer and F1 + 2 is measured. In some embodiments of any of the methods described herein, the 60 concentrations of at least two individual members of the group consisting of sCD40L, prothrombin fragment F1 + 2 and d-dimer are measured. In some embodiments of any of the methods described herein, the concentration of thrombomodulin, VCAM-1 or vWF is measured. In some embodiments of any of the methods described herein, the concentration of TNFR1 is measured. In some embodiments of any of the methods described herein, the concentrations of at least two individual members of the group consisting of IFN-γ, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP- are measured. 1, VEGF, CCL5 and IL-6. In some embodiments of any of the methods described herein, the concentration of at least one biomarker protein associated with SUHa selected from the group consisting of IFN-γ, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1 is measured , MCP-1, VEGF and IL-6. In some embodiments of any of the methods described herein, the concentration of at least one biomarker associated with SUHa selected from the group consisting of microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, 5 is measured. NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin and KIM-1. In some embodiments of any of the methods described herein, the concentration of at least one biomarker protein associated with SUHa selected from the group consisting of: CXCL10, CXCL9, IL-18, MCP-1, TNFR1, VEGF, is measured. IL-6, CCL5, IFNγ, IL-8, ICAM-1, IL-1 beta and IL-12 p70. In some embodiments of any of the methods described herein, the concentration of CXCL9, CXCL10, IL-1 beta, IL-12 p70, 10 IFN-γ, MCP-1, CCL5, sCD40L or sTNFR1 in serum is measured of the subject. In some embodiments of any of the methods described herein, the concentration of at least one biomarker associated with SUHa selected from the group consisting of microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL is measured , fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin and KIM-1 in the subject's urine. In some embodiments of any of the methods described herein, the concentration of NGAL, a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1 is measured +2, D-dimer, thrombomodulin or von Willebrand factor (vWF) in the subject's plasma. In some embodiments of any of the methods described herein, the concentration of Ba (for example, in the plasma sample obtained from the subject) is measured.

 twenty

In some embodiments of any of the methods described herein, the method requires recording the measured value or values of the concentration of at least one SUHa biomarker protein. The record may be written or in a computer readable medium. The method may also include communicating the measured value or values of the concentration of the at least one SUHa biomarker protein to the subject and / or a medical practitioner in whose charge the subject is placed. 25

In some embodiments, any of the methods described herein may include the step of administering to the subject the complement inhibitor at a higher dose or with an increased dosage frequency, in relation to the predetermined dosage schedule, if the subject does not It is sensitive to treatment with the inhibitor in the predetermined dosage schedule. 30

In some embodiments of any of the methods described herein, the complement inhibitor is administered to the subject in a predetermined dosage schedule based, in part, on the subject's body weight. For example, in the case of an anti-C5 antagonist antibody (eg eculizumab), for subjects having a body weight greater than or equal to 40 kg, the antibody can be administered to the subject for at least 7 weeks in the following program. : at least 800 mg of the antibody, once a week for four consecutive weeks; at least 800 mg of the antibody once during the fifth week; and at least 800 mg of the antibody biweekly below. In some embodiments, the antibody is administered to the subject for at least 7 weeks in the following program: at least 900 mg of the antibody, once a week for four consecutive weeks; at least 1200 mg of the antibody once during the fifth week; and at least 1200 mg of the antibody biweekly thereafter.

In some embodiments of any of the methods described herein, for subjects having a body weight less than 40 kg but greater than or equal to 30 kg, the antibody may be administered to the subject for at least 7 weeks in the following program: at least 500 mg of the antibody, once a week for two consecutive 45 weeks; at least 700 mg of the antibody once during the third week; and at least 700 mg of the antibody biweekly below. In some embodiments, the antibody is administered to the subject for at least 5 weeks in the following program: at least 600 mg of the antibody, once a week for two consecutive weeks; at least 900 mg of the antibody once during the third week; and at least 900 mg of the antibody biweekly below. fifty

In some embodiments of any of the methods described herein, the subject's body weight is less than 30 kg, but is greater than or equal to 20 kg and the antibody is administered to the subject for at least 5 weeks in the following program. : at least 500 mg of the antibody, once a week for two consecutive weeks; at least 500 mg of the antibody once during the third week; and at least 500 mg of antibody 55 biweekly below. In some embodiments, the antibody is administered to the subject for at least 5 weeks in the following program: at least 600 mg of the antibody, once a week for two consecutive weeks; at least 600 mg of the antibody once during the third week; and at least 600 mg of the antibody biweekly below.

 60

In some embodiments of any of the methods described herein, the subject's body weight is less than 20 kg, but is greater than or equal to 10 kg and the antibody is administered to the subject for at least 4 weeks in the following program. : at least 500 mg of the antibody once a week for a week; at least 200 mg of the antibody once during the second week; and at least 200 mg of the antibody biweekly below. In some embodiments, the antibody is administered to the subject for at least 4 65 weeks in the following program: at least 600 mg of the antibody once a week for a week; at least 300 mg of the antibody once during the second week; and at least 300 mg of the antibody biweekly below.

In some embodiments of any of the methods described herein, the subject's body weight is less than 10 kg, but is greater than or equal to 5 kg and the antibody is administered to the subject for at least 5 weeks in the following program: at least 200 mg of the antibody once a week for a week; at least 200 mg of the antibody once during the second week; and at least 200 mg of the antibody once every three weeks thereafter. In some embodiments, the antibody is administered to the subject for at least 5 weeks in the following program: at least 300 mg of the antibody, once a week for a week; at least 300 mg of the antibody once during the second week; and at least 300 mg of the antibody every three 10 weeks thereafter. Additional exemplary anti-C5 antibody dosing schedules (eg, chronic dosing schedules) for SUHa are described in International Patent Application Publication No. WO 2010/054403 (for example, Tables 1 and 2 of WO 2010 / 054403).

In some embodiments of any of the methods described herein, the inhibitor is an antibody or an antigen-binding fragment thereof, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic or an aptamer. In some embodiments, the inhibitor may be one that inhibits one or more of the complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, Factor D, Factor B, properdin, MBL, MASP-1 , MASP-2, or biologically active fragments of any of the above. In some embodiments of any of the methods described herein, the complement inhibitor inhibits one or both of the generation of anaphylactic activity associated with C5a and / or the assembly of the membrane attack complex associated with C5b.

The compositions may also contain naturally occurring or soluble forms of complement inhibitor compounds such as CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, 25 complestatin and K76 COOH.

In some embodiments, the complement inhibitor may be a complement factor 2 (CR2) H (FH) molecule comprising: a) a CR2 part comprising CR2 (eg, human CR2) or a fragment thereof, and b) an FH part comprising an FH or a fragment thereof, in which the CR2-FH molecule or fragment thereof is capable of binding with a CR2 ligand, and in which the CR2-FH molecule is able to inhibit the activation of the complement of the alternative route. Exemplary CR2-FH fusion proteins are described and exemplified, for example, in International Patent Application Publications No. WO 2007/149567 and WO 2011/143637. In some embodiments, the complement inhibitor comprises a targeting domain such as CR2 or an anti C3d antibody as described, for example, in International Patent Application Publication No. WO 2011/163412. Mergers of address domains with other complement inhibitors such as CD59, CD55 and H-type molecules can be used in the methods described herein as a complement inhibitor. See WO 2011/163412, above.

In some embodiments of any of the methods described herein, the complement inhibitor is an antagonistic antibody or antigen binding fragment thereof. The antibody or antigen binding fragment thereof can be selected from the group consisting of a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, an immunized antibody, a completely human antibody, a single chain antibody. , an Fv fragment, an Fd fragment, a Fab fragment, a Fab 'fragment and an F (ab') 2 fragment. Four. Five

In some embodiments of any of the methods described herein, the antagonist antibody is an anti C5 antibody such as eculizumab. In some embodiments, the antagonist antibody is pexelizumab, a C5 binding fragment of the anti C5 antibody.

 fifty

In some embodiments of any of the methods described herein, the complement inhibitor is selected from the group consisting of MB 12/22, MB12 / 22-RGD, ARC187, ARC1905, SSL7 and OmCI.

In some embodiments of any of the methods described herein, the subset of biomarker proteins associated with HUS from which a practitioner can determine the concentration of one or more (for example, two, three, four, five, six, seven, eight, nine, 10 or more) can be: Ba, thrombomodulin, VCAM-1, TNFR1, F1 + 2, D-dimer, CXCL10, IL-6, clusterin, TIMP-1, FABP-1, β2M and cystatin C.

The disclosure has a matrix comprising a plurality of binding agents, in which each binding agent of the plurality has a unique address in the matrix, in which the matrix does not comprise more than 500 60 unique addresses, in which each plurality binding agent binds with a different biological analyte protein and in which the matrix comprises binding agents that bind with four or more analyte proteins set forth in Table 1, for example, selected from the group consisting of : a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, 65 MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C , NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9 , KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5. The matrix is useful in any of the methods described herein. The matrix can be a protein microplate. In some embodiments, each direction of the matrix is a well of a test plate. Each matrix address can be a particle (eg, a bead) that has a binding agent immobilized therein. 5

As used herein, the term "binding agent" includes any agent of natural, synthetic or genetically engineered origin, such as a protein, that binds with an antigen (eg, a biomarker protein of SUHa) . Binding agents can be or derived from naturally occurring antibodies. A protein or binding agent can act similarly to an antibody by binding with a specific antigen to form a complex. Binding agents or proteins may include antigen binding fragments isolated from antibodies.

The matrix may comprise antibodies that bind with at least two (for example, at least three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) of analyte proteins. For example, the matrix can comprise binding agents / antibodies that bind with at least two (eg, three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a proteolytic fragment of complement component factor B (e.g., Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin , cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1). twenty

In some embodiments of the matrix disclosed herein, the matrix does not comprise more than 200 (for example no more than 175, 150, 125, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30 , 25 or 20) unique addresses.

The disclosure presents a diagnostic kit comprising one or more of any of the matrices described in this document and, optionally, instructions for (a) obtaining and / or processing a biological sample (eg, a biological liquid) of a subject and / or (b) measure one or more analytes in a biological sample (for example, a biological liquid) of a subject.

The disclosure presents a diagnostic kit comprising: (a) a test plate and (b) at least three binding agents, each binding agent being capable of binding with a different biological analyte, in which the analytes are the represented in Table 1, for example, selected from the group consisting of: a proteolytic fragment of the complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, component of complement C5a, microglobulin β2 35 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, growth factor Vascular endothelial cell (VEGF), IL-6, albumin, IL-8 and CCL5. The diagnostic kit may comprise one or more means to measure the activity of vWF in human plasma.

The disclosure presents a method to diagnose that a subject has, or is at risk of developing, atypical hemolytic uremic syndrome (SUHa). The method includes: measuring in a biological liquid the concentration of at least two biomarker proteins associated with SUHa selected from the group consisting of: a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9) , thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, component of complement C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5. The biological fluid is one obtained from a subject that is suspected of having or is at risk of developing SUHa. According to the methods, a high concentration, compared to the concentration in a normal control biological liquid of the same type, of at least one of Ba, sC5b-9, C5a, sCD40L, prothrombin fragment F1 + 2, d -dimer, thrombomodulin, VCAM-1, vWF, FABP-1, β2M, clusterin, cystatin C, TIMP-1, albumin, NGAL, CXCL10, CXCL9, IL-18, TNFR1, VCAM-1, MCP-1, VEGF, CCL5, IL-6 or IFNγ, indicates that the subject has, or is at risk of developing, SUHa. The at least two biomarkers associated with SUHa can be selected from Table 11, that is, at least two (eg, three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a fragment proteolytic factor of complement component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1).

As used herein, the term "normal", when used to modify the term "individual" or "subject" refers to an individual or group of individuals who does not have a particular disease or condition (for example, SUHa) and is not suspected of having or being at risk of developing the disease or condition. The term "normal" is also used herein to qualify a biological test sample or sample (for example, a biological liquid) isolated from a normal or healthy individual or subject (or group of such subjects), for example, a "Normal control sample" or a "normal control biological liquid".

 65

The disclosure presents a method to determine if a patient experiences a first manifestation of acute atypical hemolytic uremic syndrome (SUHa). The method comprises: measuring one or both of the concentration of D-dimer (for example, the plasma concentration of D-dimer) and the concentration of fatty acid binding protein 1 (FABP-1) (for example, the concentration in urine of FABP-1), in which an increase in the concentration of d-dimer, in relation to the concentration of d-dimer in a normal control sample, and an increase in the concentration of FABP-1, in relation to With the concentration of FABP-1 in a normal control sample 5, it indicates that the patient with SUHa experiences a first manifestation of acute SUHa. The increase in one or both of D-dimer and FABP-1 can be significant increases.

The disclosure presents a method for treating atypical hemolytic uremic syndrome (SUHa), the method comprising administering to a subject who has, is suspected to have or is at risk of developing SUHa a complement inhibitor 10 (eg, a component inhibitor of complement C5) in an amount and with a frequency sufficient to effect a physiological change in at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) biomarker proteins associated with SUHa, in which the physiological change is selected from the group consisting of: (a) a concentration reduced, compared to the concentration in a sample of biological liquid of the same type 15 obtained from the subject before treatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, a proteolytic fragment or component factor of complement B (for example, Ba or Bb), C5 soluble b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, sC5b9, microglobulin β2 (β2M), clusterin, cystatin C, NAG , TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL10, CXCL9 and KIM-1; or (b) an increased concentration, in comparison with the concentration in a sample of biological liquid of the same type obtained from a subject before treatment with the CCL5 inhibitor. The at least one biomarker associated with SUHa may be selected from Table 11, that is, at least one (eg, two, three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M) , Clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1).

The disclosure presents a method for treating atypical hemolytic uremic syndrome (SUHa) using a complement inhibitor in a manner sufficient to induce a physiological change in at least two biomarker proteins associated with SUHa. The method includes: (a) determining the concentration of at least two biomarkers associated with SUHa in a biological liquid obtained from the subject, in which the biomarker proteins associated with SUHa are selected from the group consisting of: CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d-dimer, thrombomodulin, VCAM-1 , von Willebrand factor (vWF), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9 , KIM-1 and CCL5; and (b) administer to a subject who has, is suspected of having, or is at risk of developing SUHa, a complement inhibitor in an amount and with sufficient frequency to cause a physiological change in at least each of two (2 ) biomarker proteins associated with SUHa, in which the physiological change is selected from the group consisting of: (a) a reduced concentration, compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of at least one of CXCL10, MCP-1, TNFR1, IFN-γ, IL-6, a proteolytic fragment of complement component component B (eg, Ba or Bb), soluble C5b9 (sC5b9), fragment of prothrombin F1 + 2, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, binding protein to fatty acids 1 (FABP-1), albumin, CXCL9 or KIM-1; and (b) an increased concentration in a biological liquid obtained from the subject, as compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the inhibitor, of CCL5. The method may also include determining if physiological changes have occurred. The at least two biomarkers associated with SUHa can be selected from Table 11, that is, at least two (eg, three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a fragment proteolytic factor of complement component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, 50 thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1).

The methods may further include the step of measuring the concentrations of at least two individual biomarker proteins associated with SUHa in a biological liquid, in which the biomarker proteins associated with SUHa are selected from the group consisting of: a proteolytic fragment of the component factor of complement B (for example, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer , CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL , fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5. The biological fluid is obtained from the subject. The at least two biomarkers associated with SUHa can be selected from Table 11, that is, at least two (eg, three, four, five, six, seven, eight, nine, 10, 11, 12 or 13) of a fragment proteolytic factor of complement component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, microglobulin β2 (β2M), clusterin , cystatin C, 65 TIMP-1 and fatty acid binding protein 1 (FABP-1).

Any of the methods described herein may include determining whether at least two have occurred (for example, at least three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) physiological changes. In some embodiments, the concentrations of at least two IFN-γ, ICAM-1, IL-1 beta and IL-12 p70 are reduced. In some embodiments, the concentrations of both Ba and sC5b9 are reduced. In some embodiments, the concentration (for example, the concentration in urine) of 5 each of C5a and sC5b9 is reduced. In some embodiments of any of the methods described herein, the concentrations (e.g., urine concentration) of at least two (e.g., at least three, four, five, six or all) of β2M are reduced, clusterina, cystatin C, NAG, TIMP-1, NGAL and FABP-1. In some embodiments, concentrations (eg, urine concentration) of CXCL10, CXCL9 and / or KIM-1 are reduced. In some embodiments, concentrations (eg, plasma concentration) of one or both of D-dimer and F1 + 2 are reduced. In some embodiments, concentrations (eg, serum and / or plasma concentrations) of at least two (for example, at least three, or all) of sCD40L, prothrombin fragment F1 + 2 and D-dimer are reduced. In some embodiments, the concentrations of thrombomodulin, VCAM-1 and / or vWF are reduced. In some embodiments, concentrations (eg, serum concentrations) of CXCL10, MCP-1 and TNFR1 are reduced. In some embodiments, concentrations (for example, serum concentrations) of at least two (for example, at least three, four or all) of IFN-γ, ICAM-1, IL-1 beta and IL- are reduced 12 p70. In some embodiments, the at least two physiological changes may be a reduction in the concentration of at least two biomarkers associated with SUHa selected from Table 11, that is, at least two (eg, three, four, five, six, seven , eight, nine, 10, 11, 12 or 13) of a proteolytic fragment of complement component component B (for example, Ba or Bb), soluble C5b9 (sC5b9), C5a, thrombomodulin, 20 VCAM-1, prothrombin fragment F1 + 2, D-dimer, sTNFR1, β2 microglobulin (β2M), clusterin, cystatin C, TIMP-1 and fatty acid binding protein 1 (FABP-1).

In some embodiments of any of the methods described herein, the concentration of Ba (eg, concentration of Ba in plasma) is reduced by at least 10% at week 6 after the start of treatment. In some embodiments of any of the methods described herein, the concentration of Ba (eg, concentration of Ba in plasma) is reduced by at least 30% at week 12 after the start of treatment. In some embodiments of any of the methods described herein, the concentration of C5a (eg, concentration of C5a in urine) is reduced by at least 40% at week 3 after the start of treatment. In some embodiments of any of the methods described herein, the concentration of C5a (eg, concentration of C5a in urine) is reduced by at least 70% at week 6 after the start of treatment. In some embodiments of any of the methods described herein, the concentration of C5b-9 (eg, concentration of C5b-9 in urine or plasma) is reduced by at least 50% at week 3 after the start of treatment. . In some embodiments of any of the methods described herein, the concentration of F1 + 2 (for example, the plasma concentration of F1 + 2) is reduced by at least 20% at week 6 after the start of treatment. . In some embodiments of any of the methods described herein, the d-dimer concentration (eg, the d-dimer plasma concentration) is reduced by at least 40% at week 6 after the start of treatment. In some embodiments of any of the methods described herein, the thrombomodulin concentration (eg, the thrombomodulin serum concentration) is reduced by at least 20% at week 12 after the start of treatment. In some embodiments of any of the methods described herein, the concentration of VCAM-1 (for example, the serum concentration of VCAM-1) is reduced by at least 20% at week 12 after the start of treatment.

In some embodiments of any of the methods described herein, the complement inhibitor is administered to the subject in an amount and with a frequency sufficient to effect a physiological change in three or more biomarkers associated with SUHa. In some embodiments, the complement inhibitor is administered to the subject in an amount and with a frequency sufficient to effect a physiological change in at least four biomarkers associated with SUHa. In some embodiments, the complement inhibitor is administered to the subject in an amount and with a frequency sufficient to effect a physiological change in at least five biomarkers associated with SUHa. In some embodiments, the complement inhibitor is administered to the subject in an amount and with a frequency sufficient to effect a physiological change in at least 10 biomarkers associated with SUHa. In some embodiments, the complement component C5 inhibitor is administered to the subject in an amount and with a frequency sufficient to effect a physiological change in 15 or more biomarkers associated with SUHa. 55

In some embodiments, a physiological change in at least two (for example, at least three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24 or 25 or more) biomarker proteins associated with SUHa are produced in two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, six weeks, two months, nine weeks or three months or more after 60 administration (eg chronic administration) of the inhibitor.

In some embodiments, the concentration of at least one is reduced (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) biomarker protein associated with SUHa by at least 5 (for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65 or 70%) 65 after administration of the inhibitor.

In some embodiments, the concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) biomarker protein associated with SUHa is reduced to within 50 (for example, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39 , 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1)% of the normal concentration of the biomarker protein after administration of one or more doses of the inhibitor. 5

In some embodiments of any of the methods described herein, the concentration of FABP-1 (e.g., FABP-1 in urine) is reduced by at least 80% (e.g., 85, 90, 95 or up to 100% ) after administration of a human complement inhibitor (for example, an anti C5 antibody). In some embodiments of any of the methods described herein, the concentration of cystatin C (for example, cystatin C in urine) is reduced by at least 80% (e.g., 85, 90, 95, 99 or up to 100 %) after administration of a human complement inhibitor (for example, an anti C5 antibody). In some embodiments of any of the methods described herein, the concentration of clusterin (e.g., clusterin in urine) is reduced by at least 80% (e.g., 85, 90, 95, 98 or up to 100%) after of the administration of a human complement inhibitor (for example, an anti-C5 antibody). In some embodiments of any of the methods described herein, the concentration of a proteolytic fragment of factor B (e.g., Ba) is reduced by at least 10% (e.g., 15, 20, 25, 30 or 40 %) after administration of a human complement inhibitor (for example, an anti-C5 antibody). In some embodiments of any of the methods described herein, the concentration of sTNFR1 is reduced by at least 80% (for example, 85, 90 or more%) after administration of a human complement inhibitor 20 (for example , an anti C5 antibody). In some embodiments of any of the methods described herein, the concentration of thrombomodulin or sVCAM-1 is reduced by at least 80% (eg, 85, 90, 95 or up to 100%) after administration of an inhibitor. of the human complement (for example, an anti C5 antibody). In some embodiments of any of the methods described herein, the concentration of one or both of F1 + 2 or D-dimer is reduced by at least 80% (for example, 85, 90, 95 or more%) after of the administration of a human complement inhibitor (for example, an anti C5 antibody).

In some embodiments of any of the methods described herein, the concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 30 18, 19, 20, 21, 22, 23, 24 or 25) of the biomarker proteins associated with SUHa is normalized after administration of the inhibitor. In some embodiments, concentrations (for example, urine concentrations) of at least three of microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP) are normalized -1), CXCL10, CXCL9 and KIM-1.

 35

As used herein, the term "normalized" or similar grammatical terms, when used in the context of the effect of a complement inhibitor therapy on the concentration or activity of a biomarker protein of SUHa, refers to a concentration or activity measured in a biological liquid of a biomarker protein that has been brought into 50 (for example, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 40 or 1)% of the average concentration or range of activity of the SUHa biomarker protein as measured in a sample of the same type of biological liquid obtained of a group of healthy individuals (normal individuals). For example, treating a patient with SUHa with a complement inhibitor can normalize a high urine clusterin concentration up to, for example, 20% of the average average urine clusterin concentration range. In some embodiments, treatment with the complement inhibitor 45 would restore clusterin urine concentration to within the normal average clusterin urine concentration range.

In some embodiments of any of the methods described herein, the subject has received dialysis at least once (for example, at least twice, three, four or five times or more) in the three months (for example, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 weeks) before treatment with the inhibitor. For example, in some embodiments, the subject received dialysis once two months before receiving complement inhibitor therapy. In another example, the subject may be one who has received dialysis three times in the three month period just before receiving complement inhibitor therapy. In some embodiments of any of the methods described herein, in relation to the concentration in a healthy subject, the concentrations of one or more of TNFR1, Ba, the thrombomodulin fragment F1 + 2 and sC5b9 are elevated. In some embodiments of any of the methods described herein, in relation to concentrations (e.g., urine concentrations) in a healthy human being, the concentrations of one or more of β2M, sC5b9, C5a, cystatin C, clusterina, TIMP-1 and NGAL are elevated.

 60

In some embodiments of any of the methods described herein, the subject (eg, a human subject) experiences a first manifestation of acute SUHa. For example, prior to treatment with the complement inhibitor, the subject may have elevated concentrations, relative to normal concentrations, of one or both of D-dimer and FABP-1.

 65

In some embodiments of any of the methods described herein, the subject (for example, a human subject) is one who has SUHa, but is considered to be in clinical remission (for example, the subject is one that has normal levels of platelets or other hematological markers such as LDH or haptoglobin). In some embodiments, said subject is one that has elevated levels of one or more of the SUHa biomarkers described herein, including, but not limited to, one or more of Ba, D-dimer, VCAM-1 and prothrombin fragments. 1 + 2 5

It is understood that for any of the methods described herein, the concentration and / or activity of one or more SUHa biomarker proteins can be determined. For example, in some embodiments, a practitioner can measure the activity of vWF in a biological sample obtained from the subject as a representation of the concentration of vWF (or other biomarker proteins) in the sample. Methods for evaluating the relative activity of SUHa biomarker proteins set forth in Table 1 are known in the art.

As discussed in detail in this document (for example, in the work examples), SUHa is a life-threatening, genetic disease that involves chronic complement deregulation. Patients suffering from the disease suffer, among other things, thrombotic microangiopathy (TMA), which can result in stroke and kidney failure. It has been shown that eculizumab, an anti-C5 antagonist antibody, dramatically reduces TMA, normalizes platelet levels and improves renal function in patients with SUHa. Even so, even with the clear and robust clinical benefit of complement inhibitor therapy for SUHa patients, some patients still experience elevated levels of several SUHa biomarker proteins compared to treatment. For example, the inventors have found that, in some patients, the levels of a proteolytic fragment 20 of the complement component component B (for example, Ba or Bb) (for example, in plasma) do not normalize after treatment with an antibody anti C5 antagonist. In addition, for some patients, the levels of prothrombin fragment 1 + 2, D-dimer, thrombomodulin, VCAM-1, TNFR1 and CXCL10 are reduced but not normalized over time. Although the disclosure is not linked to any particular theory or mechanism of action, these observations suggest that, for some patients, low levels of inflammation and coagulopathy may persist even with complement inhibitory therapy. Therefore, the disclosure contemplates methods in which a complement inhibitor is administered in combination with a second therapy to address the low level of persistent inflammation in some patients with SUHa.

Therefore, in another aspect, the disclosure presents a method for treating atypical hemolytic uremic syndrome 30 (SUHa). The method comprises administering (for example, administering chronically) to a subject (eg, a human subject) who has, is suspected to have, or is at risk of developing, SUHa a therapeutically effective amount of a complement inhibitor ( for example, an inhibitor of complement component C5) and a therapeutically effective amount of: (i) an anticoagulant, (ii) a fibrinolytic agent; (iii) an anti-inflammatory agent; or (iv) an inhibitor of IL-6, IL-8, CXCL-9, IL-18 or VEGF. In some embodiments, two complement inhibitors can be used (for example, a C5 inhibitor and a C3 inhibitor, such as an anti Factor B antibody, an anti C3 antibody or an anti C3b antibody). In some embodiments, at the time of stopping therapy with a C5 inhibitor, a C3 complement component inhibitor may be administered to the patient for a time sufficient to reduce upstream alternative route activation.

 40

In some embodiments, the methods described herein may include monitoring the status of one or more SUHa biomarkers and determining whether to initiate a second therapy (in addition to complement inhibitory therapy) or modify the dosage regimen of one or more second therapies that are given to a patient with SUHa. For example, during treatment (for example, chronic treatment) with a complement inhibitor, the concentration of one or more biomarker proteins associated with SUHa can be measured in one or more biological liquids obtained from the subject. If the concentration of one or more of the biomarker proteins has not normalized and / or remains high, a medical practitioner may choose to administer to the subject one or more additional side agents (eg, anti-inflammatory) to address any pathophysiological effect resulting from the elevated biomarkers.

 fifty

The complement inhibitor may be any of those described herein. In some embodiments of any of the methods described herein, the inhibitor is antibody or an antigen binding fragment thereof, a small molecule, a polypeptide, a polypeptide analog, a peptidomimetic or an aptamer. In some embodiments, the inhibitor may be one that inhibits one or more of the complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, Factor D, Factor B, properdin, MBL, MASP-1 , 55 MASP-2 or biologically active fragments of any of the above. In some embodiments of any of the methods described herein, the complement inhibitor inhibits one or both of the generation of anaphylactic activity associated with C5a and / or the assembly of the membrane attack complex associated with C5b.

 60

The compositions may also contain naturally occurring or soluble forms of complement inhibitor compounds such as CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin and K76 COOH.

In some embodiments of any of the methods described herein, the complement inhibitor is an antagonistic antibody or antigen binding fragment thereof. The antibody or antigen-binding fragment thereof can be selected from the group consisting of a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, an immunized antibody, a human supplemented antibody, a single chain antibody. , an Fv fragment, an Fd fragment, a Fab fragment, a Fab 'fragment and an F (ab') fragment

 5

In some embodiments of any of the methods described herein, the antagonist antibody is an anti-C5 antibody such as eculizumab. In some embodiments, the antagonist antibody is pexelizumab, a C5 binding fragment of anti-C5 antibody.

In some embodiments of any of the methods described herein, the complement inhibitor is selected from the group consisting of MB 12/22, MB12 / 22-RGD, ARC187, ARC1905, SSL7 and OmCI.

In some embodiments of any of the methods described herein, the anticoagulant is selected from the group consisting of: a coumarin, heparin, a factor Xa inhibitor and a thrombin inhibitor. Examples of anticoagulants include, for example, warfarin (Coumadina), aspirin, heparin, fenindione, fondaparinux, idraparinux and thrombin inhibitors (for example, argatroban, lepirudin, bivalirudin or dabigatran).

In some embodiments of any of the methods described herein, the fibrinolytic agent is selected from the group consisting of ancrod, ε-aminocaproic acid, antiplasmin a1, prostacyclin and defibroth.

 twenty

In some embodiments of any of the methods described herein, the anti-inflammatory agent is an anti-cytokine agent such as an antagonistic antibody (or antigen-binding fragment thereof) or a soluble cytokine receptor, which binds with a inflammatory cytokine and inhibits the activity of cytokine. The anti-cytokine agent can be, for example, a TNF inhibitor (for example, an anti-TNF antibody or soluble TNF receptor protein) or an anti-CD20 agent. 25

Anti-inflammatory agents also include, for example, steroids (for example, dexamethasone), non-steroidal anti-inflammatory drugs (NSAIDs) (for example, indomethacin, naproxen, sulindac, diclofenac, aspirin, flurbiprofen, oxaprozin, salsalate, difunisal, piroxicam, etoladodom, eto meclofenamate, ibuprofen, fenoprofen, ketoprofen, nabumetone, tolmetin, magnesium choline salicylate, COX-2 inhibitors, TNF alpha 30 antagonists (etanercept, adalimumab, infliximab, golimumab), disease modifying anti-rheumatic drugs (FARME) (for example, sulfasalazine, methotrexate), cyclosporine, retinoids and corticosteroids.

The disclosure presents a method to determine if the concentration of one or more biomarker proteins associated with SUHa is high in a patient who has, is suspected of having or is at risk of developing, atypical hemolytic uremic syndrome (SUHa), in which The method comprises: (i) measuring in a biological sample obtained from the patient the concentration of each of at least two biomarkers associated with SUHa in Table 11 (subsequently), that is, selected from the group consisting of: a proteolytic fragment of the factor B, C5a, soluble C5b-9 (sC5b-9), soluble TNFR1 (sTNFR1), soluble VCAM-1 (sVCAM-1), thrombomodulin, prothrombin fragments 1 and 2 (F1 + 2), D-dimer, clusterin , TIMP-1, FABP-1, microglobulin beta-2 (β2m) and cystatin-C, and (ii) 40 determine if the patient has a high concentration of each of at least two of the biomarkers associated with SUHa compared to a normal control concentration thereof at minus two biomarkers. In some embodiments, the at least two biomarker proteins associated with SUHa are measured using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). The biological liquid can be, for example, blood, a blood fraction (for example, plasma or serum) or urine. It is understood that any combination of two or more (eg, three, four, five, six, seven, eight, nine, 10, 11 or 12) any of the aforementioned SUHa biomarkers can be measured and analyzed according to the methods described in this document.

The disclosure presents a method for diagnosing a patient who has atypical hemolytic uremic syndrome 50 (SUHa) (or confirming a diagnosis of SUHa), for example, in which the patient has met two or more of the inclusion criteria analyzed in the Example 1), in which the method comprises: (1) measuring in a biological sample obtained from a patient suspected of having SUHa or at risk of developing SUHa the concentration of each of at least two biomarkers associated with SUHa selected from the group consisting of: a proteolytic fragment of factor B, C5a, soluble C5b-9 (sC5b-9), soluble TNFR1 (sTNFR1), soluble VCAM-1 (sVCAM-1), thrombomodulin, prothrombin fragments 1 and 2 ( F1 + 2), D-dimer, clusterin, TIMP-1, FABP-1, microglobulin beta-2 (β2m) and cystatin-C, and (ii) diagnose that a patient has SUHa (or confirm a diagnosis of SUHa) if the concentration of each of at least two of the biomarkers associated with SUHa is high compared to a normal control concentration thereof at least two biomarkers. In some embodiments, the at least two biomarker proteins associated with SUHa are measured using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). The biological fluid can be, for example, blood, a blood fraction (for example, plasma or serum) or urine. It is understood that any combination of any two or more (for example, three, four, five, six, seven, eight, nine, 10, 11 or 12) of the aforementioned SUHa biomarkers can be measured and analyzed according to the methods described in this document. 65

A normal control concentration, as used in any of the methods described herein, may be (or may be based on), for example, the concentration of a biomarker protein associated with SUHa given in a biological sample or biological samples obtained. of one or more (for example, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35 or 40 or more) healthy individuals. In some embodiments, a normal control concentration of a biomarker may be (or may be based on), for example, the concentration of the biomarker in a pooled sample obtained from two or more (eg, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35 or 40 or more) healthy individuals. In some embodiments of any of the methods described herein, the pooled samples may be from healthy individuals or, at least, individuals who do not have or are not suspected of having (are not at risk of developing) SUHa. For example, determining whether a subject is one who has SUHa may involve comparing the measured concentration of one or more complement component proteins (for example, Table 1 or Table 11) in a biological sample (or several different types of samples biological) obtained from the patient and compare the measured concentration with the average concentration of the same proteins in the pooled healthy samples. Said control concentrations of healthy humans may be, in some embodiments, a range of values, or a median of values or the average value obtained from the range. fifteen

In some embodiments, the concentration of at least one biomarker associated with SUHa is measured in two or more types of biological fluid. In some embodiments, the concentration of the first of at least two SUHa biomarker proteins is measured in one type of biological liquid and the concentration of the second of the at least two SUHa biomarker proteins is measured in a second type of liquid. twenty

In some embodiments of any of the methods described herein, the concentration of the factor B proteolytic fragment is measured. The fragment may be, for example, Ba. The biological sample can be a plasma sample. As described in Table 11, the normal control concentration of Ba may be less than 1000 ng / ml. The normal control concentration of Ba may be less than 600 ng / ml. The normal control concentration of Ba can be between 300 and 600 ng / ml.

In some embodiments, the concentration of Ba in the biological sample is considered high when it is at least twice as high as the normal control concentration of Ba. In some embodiments, the concentration of Ba in the biological sample is considered high when it is at least five times greater than the normal control concentration of Ba. In some embodiments, the concentration of Ba in the biological sample is considered high when it is at least 1500 ng / ml. In some embodiments, the concentration of Ba in the biological sample is considered high when it is at least 2500 ng / ml.

In some embodiments of any of the methods described herein, the concentration of C5a is measured. The biological sample in which C5a is measured may be a urine sample. And in some embodiments, the normal control concentration of C5a is less than 2 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of C5a is less than 1 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of C5a is between 0 and 0.7 ng per mg of creatinine in urine. 40

In some embodiments of any of the methods described herein, the concentration of C5a in the biological sample is considered high when it is at least twice as high as the normal control concentration of C5a. In some embodiments, the concentration of C5a in the biological sample is considered high when it is at least ten times greater than the normal control concentration of C5a. In some embodiments, the concentration of C5a in the biological sample is considered high when it is at least forty times greater than the normal control concentration of C5a. In some embodiments, the concentration of C5a in the biological sample is considered high when it is at least 5 ng per mg of creatinine in urine. In some embodiments, the concentration of C5a in the biological sample is considered high when it is at least 9 ng per mg of creatinine in urine. fifty

In some embodiments of any of the methods described herein, the concentration of sC5b-9 is measured. The biological sample in which sC5b-9 is measured may be a urine sample. And in some embodiments, the normal control concentration of sC5b-9 is less than 2 ng per mg of creatinine in urine. The normal control concentration of sC5b-9 may be less than 1 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of sC5b-9 is between 0 and 0.6 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of sC5b-9 in the biological sample is considered high when it is at least ten times greater than the normal control concentration of sC5b-9. In some embodiments, the concentration of sC5b-9 in the biological sample is considered high when it is at least fifty times greater than the normal control concentration of sC5b-9. In some embodiments, the concentration of sC5b-9 in the biological sample is considered high when it is at least one hundred times greater than the normal control concentration of sC5b-9. In some embodiments, the concentration of sC5b-9 in the biological sample is considered high when it is at least 20 ng per mg of 65 creatinine in urine. In some embodiments, the concentration of sC5b-9 in the biological sample is considered high when it is at least 30 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of sTNFR1 is measured. The biological sample in which sTNFR1 is measured may be a serum sample. And in some embodiments, the normal control concentration of sTNFR1 is less than 2000 pg / ml. In some embodiments, the normal control concentration of sTNFR1 is less than 1500 pg / ml. In some embodiments, the normal control concentration of sTNFR1 is between 400 and 1500 pg / ml.

In some embodiments of any of the methods described herein, the concentration of sTNFR1 in the biological sample is considered high when it is at least twice as high as the normal control concentration of sTNFR1. In some embodiments, the concentration of sTNFR1 in the biological sample is considered high when it is at least five times greater than the normal control concentration of sTNFR1. In some embodiments, the concentration of sTNFR1 in the biological sample is considered high when it is at least fifteen times greater than the normal control concentration of sTNFR1. In some embodiments, the concentration of sTNFR1 in the biological sample is considered high when it is at least 10,000 pg / ml. In some embodiments, the concentration of sTNFR1 in the biological sample is considered high when it is at least 15,000 pg / ml.

In some embodiments of any of the methods described herein, the concentration of sVCAM-1 is measured. The biological sample in which sVCAM-1 is measured may be a serum sample. And 20 in some embodiments, the normal control concentration of sVCAM-1 is less than 500 ng / ml. In some embodiments, the normal control concentration of sVCAM-1 is less than 300 ng / ml. In some embodiments, the normal control concentration of sVCAM-1 is between 100 and 500 ng / ml.

In some embodiments of any of the methods described herein, the concentration of 25 sVCAM-1 in the biological sample is considered high when it is at least 10% higher than the normal control concentration of sVCAM-1. In some embodiments, the concentration of sVCAM-1 in the biological sample is considered high when it is at least 30% higher than the normal control concentration of sVCAM-1. In some embodiments, the concentration of sVCAM-1 in the biological sample is considered high when it is at least 50% higher than the normal control concentration of sVCAM-1. In some embodiments, the concentration of sVCAM-1 in the biological sample is considered high when it is at least 550 ng / ml. In some embodiments, the concentration of sVCAM-1 in the biological sample is considered high when it is at least 650 ng / ml.

In some embodiments of any of the methods described herein, the concentration of thrombomodulin is measured. The biological sample in which thrombomodulin is measured may be a plasma sample. And in some embodiments, the normal thrombomodulin control concentration is less than 5 ng / ml. In some embodiments, the normal thrombomodulin control concentration is less than 3 ng / ml. In some embodiments, the normal thrombomodulin control concentration is between 2 and 6 ng / ml.

 40

In some embodiments of any of the methods described herein, the concentration of thrombomodulin in the biological sample is considered high when it is at least 10% greater than the normal control concentration of thrombomodulin. In some embodiments, the concentration of thrombomodulin in the biological sample is considered high when it is at least 30% higher than the normal control concentration of thrombomodulin. In some embodiments, the thrombomodulin concentration in the biological sample is considered high when it is at least 50% higher than the normal thrombomodulin control concentration. In some embodiments, the concentration of thrombomodulin in the biological sample is considered high when it is at least 8 ng / ml. In some embodiments, the concentration of thrombomodulin in the biological sample is considered high when it is at least 10 ng / ml.

 fifty

In some embodiments of any of the methods described herein, the concentration of F1 + 2 is measured. The biological sample in which F1 + 2 is measured can be a plasma sample. And in some embodiments, the normal control concentration of F1 + 2 is less than 400 pmol / l. In some embodiments, the normal control concentration of F1 + 2 is less than 300 pmol / l. In some embodiments, the normal control concentration of F1 + 2 is between 50 and 400 pmol / l. 55

In some embodiments of any of the methods described herein, the concentration of F1 + 2 in the biological sample is considered high when it is at least 30% greater than the normal control concentration of F1 + 2. In some embodiments, the concentration of F1 + 2 in the biological sample is considered high when it is at least 50% higher than the normal control concentration of F1 + 2. In some embodiments, the concentration of F1 + 2 in the biological sample is considered high when it is at least 100% higher than the normal control concentration of F1 + 2. In some embodiments, the concentration of F1 + 2 in the biological sample is considered high when it is at least 900 pmol / l. In some embodiments, the concentration of F1 + 2 in the biological sample is considered high when it is at least 1000 pmol / l.

 65

In some embodiments of any of the methods described herein, the concentration of D-dimer is measured. The biological sample in which D-dimer is measured may be a plasma sample. And in some embodiments, the normal control concentration of D-dimer is less than 500 μg / l. In some embodiments, the normal control concentration of D-dimer is less than 400 µg / l. In some embodiments, the normal control concentration of D-dimer is between 100 and 500 µg / l.

 5

In some embodiments of any of the methods described herein, the concentration of D-dimer in the biological sample is considered high when it is at least twice as high as the normal control concentration of D-dimer. In some embodiments, the concentration of D-dimer in the biological sample is considered high when it is at least five times greater than the normal control concentration of D-dimer. In some embodiments, the concentration of D-dimer in the biological sample is considered high when it is at least ten times greater than the normal control concentration of D-dimer. In some embodiments, the concentration of D-dimer in the biological sample is considered high when it is at least 1500 μg / l. In some embodiments, the concentration of D-dimer in the biological sample is considered high when it is at least 2500 μg / l.

 fifteen

In some embodiments of any of the methods described herein, the concentration of clusterin is measured. The biological sample in which clusterin is measured may be a urine sample. And in some embodiments, the normal control concentration of clusterin is less than 500 ng per mg of creatinine in urine. The normal control concentration of clusterin can be, for example, less than 400 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of clusterin is between 0 and 500 ng 20 per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of clusterin in the biological sample is considered high when it is at least twice as high as the normal control concentration of clusterin. In some embodiments, the concentration of clusterin in the biological sample is considered high when it is at least five times greater than the normal control concentration of clusterin. In some embodiments, the concentration of clusterin in the biological sample is considered high when it is at least ten times greater than the normal control concentration of clusterin. In some embodiments, the concentration of clusterin in the biological sample is considered high when it is at least 900 ng per mg of creatinine in urine. In some embodiments, the concentration of clusterin in the biological sample is considered high when it is at least 1200 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of TIMP-1 is measured. The biological sample in which TIMP-1 is measured may be a urine sample. And in some embodiments, the normal control concentration of TIMP-1 is less than 10 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of TIMP-1 is less than 5 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of TIMP-1 is between 0 and 10 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of TIMP-1 in the biological sample is considered high when it is at least twice as high as the normal control concentration of TIMP-1. In some embodiments, the concentration of TIMP-1 in the biological sample is considered high when it is at least ten times greater than the normal control concentration of TIMP-1. In some embodiments, the concentration of TIMP-1 in the biological sample is considered high when it is at least twenty times greater than the normal control concentration of TIMP-1. In some embodiments, the concentration of TIMP-1 in the biological sample is considered high when it is at least 15 ng per mg of creatinine in urine. In some embodiments, the concentration of TIMP-1 in the biological sample is considered high when it is at least 20 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of FABP-1 (also referred to herein as L-FABP-1) is measured. The biological sample in which C5a is measured may be a urine sample. And in some embodiments, the normal control concentration of FABP-1 is less than 20 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of FABP-1 is less than 15 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of FABP-1 is between 0 and 20 ng per mg of creatinine in urine. 55

In some embodiments of any of the methods described herein, the concentration of FABP-1 in the biological sample is considered high when it is at least twice as high as the normal control concentration of FABP-1. In some embodiments, the concentration of FABP-1 in the biological sample is considered high when it is at least ten times greater than the normal control concentration of FABP-1. In some embodiments, the concentration of FABP-1 in the biological sample is considered high when it is at least twenty times greater than the normal control concentration of FABP-1. In some embodiments, the concentration of FABP-1 in the biological sample is considered high when it is at least 40 ng per mg of creatinine in urine. In some embodiments, the concentration of FABP-1 in the biological sample is considered high when it is at least 50 ng per mg of creatinine in urine. 65

In some embodiments of any of the methods described herein, the concentration of β2m is measured. The biological sample in which β2m is measured may be a urine sample. And in some embodiments, the normal control concentration of β2m is less than 5 μg per mg of creatinine in urine. In some embodiments, the normal control concentration of β2m is less than 3 μg per mg of creatinine in urine. In some embodiments, the normal control concentration of β2m is between 0 and 5 μg per mg of creatinine 5 in urine.

In some embodiments of any of the methods described herein, the concentration of β2m in the biological sample is considered high when it is at least twice as high as the normal control concentration of β2m. In some embodiments of any of the methods described herein, the concentration of β2m in the biological sample is considered high when it is at least ten times greater than the normal control concentration of β2m. In some embodiments, the concentration of β2m in the biological sample is considered high when it is at least twenty times greater than the normal control concentration of β2m. In some embodiments, the concentration of β2m in the biological sample is considered high when it is at least 15 μg per mg of creatinine in urine. In some embodiments, the concentration of β2m in the biological sample is considered high when it is at least 20 μg per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of cystatin-C is measured. The biological sample in which cystatin-C is measured may be a urine sample. And in some embodiments, the normal control concentration of cystatin-C is less than 400 ng per mg of urine creatinine. In some embodiments, the normal control concentration of cystatin-C is less than 300 ng per mg of creatinine in urine. In some embodiments, the normal control concentration of cystatin-C is between 0 and 400 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentration of cystatin-C in the biological sample is considered high when it is at least twice as high as the normal control concentration of cystatin-C. In some embodiments, the concentration of cystatin-C in the biological sample is considered high when it is at least ten times greater than the normal control concentration of cystatin-C. In some embodiments, the concentration of cystatin-C in the biological sample is considered high when it is at least twenty times greater than the normal control concentration of cystatin-C. In some embodiments, the concentration of cystatin-C in the biological sample is considered high when it is at least 900 ng per mg of creatinine in urine. In some embodiments, the concentration of cystatin-C in the biological sample is considered high when it is at least 1200 ng per mg of creatinine in urine.

In some embodiments of any of the methods described herein, the concentrations of two or more of the proteolytic fragments of factor B, C5a and sC5b-9 are measured. In some embodiments of any of the methods described herein, the concentrations of C5a and sC5b-9 are measured. In some embodiments of any of the methods described herein, the concentrations of sVCAM-1 and thrombomodulin are measured. In some embodiments of any of the methods described herein, the concentrations of F1 + 2 and D-dimer are measured. In some embodiments of any of the methods described herein, the concentrations of two or more clusterin, TIMP-1, β2m, FABP-1 and cystatin-C are measured.

The disclosure presents a method to evaluate the level of alternative route activation in a patient who has SUHa, is suspected of having SUHa or is at risk of developing SUHa, before, during or after treatment with a complement inhibitor, such as an anti-C5 antibody. The method comprises: measuring the concentration of a proteolytic fragment of factor B (for example, Ba or Bb) in a biological sample obtained from a patient treated with a complement inhibitor (for example, an inhibitor of the human complement component C5, such as an anti-C5 antibody).

 fifty

The disclosure presents a method to determine if a patient has responded to therapy with a complement inhibitor (for example, has had a reduction in the risk of developing thrombosis or has had a reduction in the number, frequency or occurrence of thrombotic microangiopathy), the method comprising measuring the concentration of one or more thrombosis or coagulation biomarkers set forth in Table 1 or 11, for example, F1 + 2, D-dimer, vWF or thrombomodulin, in a biological sample obtained from a patient at high risk of , 55 who suffers or is suspected of having thrombotic microangiopathy (TMA) and treated with a complement inhibitor; and determining that the patient has responded to therapy if the concentration of the biomarker (s) in the biological sample is reduced, compared to the concentration of the biomarker (s) in a biological sample of the same type obtained from the patient before treatment with the inhibitor of the complement or determine that the patient has not responded to therapy if the concentration of the biomarker (s) in the biological sample is not reduced, compared to the concentration of the biomarker in a biological sample of the same type obtained from the patient before treatment with the complement inhibitor. In some embodiments, the patient has, is suspected of having or is at risk of developing SUHa.

The disclosure presents a method to determine if a patient with SUHa has responded to therapy with a complement inhibitor, the method comprising measuring the concentration of one or more terminal complement activation biomarkers set forth in Table 1 or 11, for example , C5a and / or sC5b-9, in a biological sample obtained from a patient who has, is suspected of having or is at risk of developing SUHa and treated with a complement inhibitor (for example, an anti-C5 antibody); and determine that the patient has responded to the therapy if the concentration of the biomarker (s) in the biological sample is reduced, compared to the concentration of the biomarker (s) in a biological sample of the same type obtained from the patient before treatment with the inhibitor. of complement or determine that the patient has not responded to therapy if the concentration of the biomarker (s) in the biological sample is not reduced, compared to the concentration of the biomarkers in a biological sample of the same type obtained from the patient before treatment with the complement inhibitor. Therefore, the method can be used to evaluate or monitor terminal complement blockage in a patient with SUHa treated with a complement inhibitor. In embodiments in which the patient is not sensitive, or is less sensitive to therapy, the method may also include changing the dose amount or dose frequency of the complement inhibitor or choosing a different complement inhibitor (e.g., a C3 activation inhibitor) for use in the treatment of the patient.

The disclosure presents a method to determine if a patient with SUHa has responded to therapy with a complement inhibitor, the method comprising measuring the concentration of one or more biomarkers of vascular inflammation or endothelial activation set forth in Table 1 or 11, by for example, sTNFR1, sVCAM-1, or thrombomodulin, in a biological sample obtained from a patient who has, is suspected of having or is at risk of developing SUHa; and determine that the patient has responded to therapy if the concentration of the biomarker (s) in the biological sample is reduced, compared to the concentration of the biomarker (s) in a biological sample of the same type obtained from the patient before treatment with the inhibitor of complement or determine that the patient has not responded to therapy if the concentration of the biomarker (s) in the biological sample is not reduced, compared to the concentration of the biomarkers in a biological sample of the same type obtained from the patient before treatment with the complement inhibitor. Therefore, the method can be used to evaluate or monitor vascular inflammation in a patient with SUHa treated with a complement inhibitor. In embodiments in which the patient is not sensitive, or is less sensitive to therapy, the method may also include changing the dose amount or dose frequency of the complement inhibitor or choosing a different complement inhibitor (e.g., an inhibitor of the activation of C3) for use in the treatment of the patient.

 30

The disclosure presents a method to determine if a patient with SUHa has responded to therapy with a complement inhibitor, the method comprising measuring the concentration of one or more biomarkers of renal lesion set forth in Table 1 or 11, for example, clusterin, TIMP-1, FABP-1, β2m and / or cystatin-C, in a biological sample obtained from a patient who has, is suspected of having or is at risk of developing SUHa; and determining that the patient has responded to the therapy if the concentration of the biomarker (s) in the biological sample is reduced, compared with the concentration of the biomarker (s) in a biological sample of the same type obtained from the patient before treatment with the inhibitor of complement or determine that the patient has not responded to therapy if the concentration of the biomarker (s) in the biological sample is not reduced, compared to the concentration of the biomarker (s) in a biological sample of the same type obtained from the patient before treatment with the complement inhibitor. Therefore, the method can be used to evaluate or monitor renal injury in a patient with SUHa treated with a complement inhibitor. In embodiments in which the patient is not sensitive, or is less sensitive to therapy, the method may also include changing the dose amount or dose frequency of the complement inhibitor or choosing a different complement inhibitor (e.g., an inhibitor of activation of C3) for use in the treatment of the patient.

 Four. Five

The inventors have also discovered that, in patients with SUHa, the relative increase in concentrations of C5a and sC5b-9 terminal complement activation markers (for example, urine concentrations) is much greater than the relative increase in marker levels of activation of alternative complement pathway (for example, Ba) in these patients. That is, the median concentration of C5a and sC5b-9 in patients with SUHa was 45 and 305 times higher, respectively, than the median concentration of these 50 markers in normal healthy humans, while the median concentration of Ba was only about 5 times higher than the median of the concentration of Ba in healthy normal humans. Although not linked to any particular theory or mechanism of action, the inventors believe that the activation ratio of the terminal complement to alternative route activation is a useful diagnostic tool for SUHa. Therefore, in another aspect, the disclosure presents a method for diagnosing SUHa or confirming a diagnosis of SUHa, including said method comparing the level of terminal complement activation (for example, sC5b-9 or C5a) with the level of activation. of the upstream alternative route activation (for example, Ba or Bb) (in relation to normal healthy humans), in which a higher degree of terminal activation in relation to the alternative route activation is an indication that the patient It has SUHa. For example, an indicative ratio of SUHa could be, for example, approximately 45: 5 or 305: 5, induction factor of activation of the terminal complement with respect to induction factor of alternative route activation. In addition, the inventors believe that this relationship may be useful for distinguishing SUHa from other diseases associated with complement, such as, for example, thrombotic thrombocytopenic purpura (TTP), which may not show such difference in terminal complement and alternative route activation levels. Upstream.

 65

"Polypeptide", "peptide" and "protein" are used interchangeably and mean any chain with peptide linkages of amino acids, regardless of length or post-translational modification. The proteins described herein may contain or be wild-type proteins or may be variants that are not more than 50 (for example, not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40 or 50) conservative amino acid substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; 5 asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine.

As used herein, the percentage (%) of amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and entering gaps. , if necessary, to achieve 10 percent maximum sequence identity. Alignment can be achieved for the purpose of determining the percentage of sequence identity in various ways that are within the skill of the art, for example, using publicly available computer software such as BLAST software. Appropriate parameters for measuring alignment can be determined, including any algorithm necessary to achieve maximum alignment over the full length of the sequences that are compared by known methods. fifteen

As used herein, the term "antibody" includes both complete antibodies and antigen-binding fragments of complete antibodies. Complete antibodies include different antibody isotypes including IgM, IgG, IgA, IgD and IgE antibodies. The term "antibody" includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primed antibody, a deimmunized antibody and a fully human antibody. The antibody can be prepared in or obtained from any of a variety of species, for example, mammals such as humans, non-human primates (eg, orangutans, baboons or chimpanzees), horses, cows, pigs, sheep, goats, dogs, Cats, rabbits, guinea pigs, gerbils, hamsters, rats and mice. The antibody can be a purified antibody or a recombinant one. 25

As used herein, the term "antibody fragment", "antigen-binding fragment" or similar expressions refers to a fragment of an antibody that retains the ability to bind with a target antigen (eg, human C5 ) and inhibit the activity of the target antigen. Such fragments include, for example, a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab ’fragment or an F (ab’) 2 fragment. An scFv fragment is a single polypeptide chain that includes both the light chain and heavy chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, for example, Todorovska et al. (2001) J Immunol Methods 248 (1): 47-66; Hudson and Kortt (1999) J Immunol Methods 231 (1): 177-189; Poljak (1994) 35 Structure 2 (12): 1121-1123; and Rondon and Marasco (1997) Annual Review of Microbiology 51: 257-283. The term "antibody" also encompasses bispecific antibodies (including DVD-Ig antibodies; see below). Bispecific antibodies are monoclonal antibodies, preferably human or humanized, which have binding specificities for at least two different antigens.

 40

As used herein, the term "antibody" also includes, for example, single domain antibodies such as camelized single domain antibodies. See, for example, Muyldermans et al. (2001) Trends Biochem Sci 26: 230-235; Nuttall et al. (2000) Curr Pharm Biotech 1: 253-263; Riechmann et al. (1999) J Immunol Meth 231: 25-38; PCT application publications No. WO 94/04678 and WO 94/25591; and U.S. Patent No. 6,005,079. In some embodiments, the disclosure provides antibodies of a single domain comprising two VH domains with modifications such that antibodies of a single domain are formed.

Unless defined otherwise, all the technical and scientific terms used in this document have the same meaning as a regular expert in the field to which this disclosure belongs. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the methods and compositions disclosed herein.

Other elements and advantages of the present disclosure, for example, methods for treating disorders associated with complement in a subject, will be apparent from the following description, examples and claims.

Brief description of the drawings

Fig. 1A is a dot plot depicting the concentration of C5a (in ng / mg of urine creatine) in the urine of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after onset of treatment with eculizumab. The concentration of C5a in urine was also measured in the urine of normal, healthy individuals (NORM).

Fig. 1B is a point diagram representing the concentration of sC5b-9 (in ng / mg of urine creatine) 65 in the urine of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks later of the start of treatment with eculizumab. The concentration of C5b9 in urine in the urine of normal, healthy individuals (NORM) was also measured.

Fig. 1C is a point diagram representing the concentration of complement component Ba (in ng / ml) in the plasma of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several 5 weeks after the onset of Eculizumab treatment. The concentration of Ba in the plasma of normal, healthy (normal) individuals was also measured.

Fig. 1D is a bar graph that represents the average percentage (%) of reduction of urine C5a levels (Y axis) over time in patients with SUHa (N = 26) after the start of treatment with 10 eculizumab. The x-axis indicates the week of the visit of the SUHa patient for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation at week 3 after the start of treatment.

Fig. 1E is a bar graph that represents the average percentage (%) of reduction of sC5b-15 levels in urine (Y axis) over time in patients with SUHa (N = 23) after onset of treatment with eculizumab. The x-axis indicates the week of the visit of the patient with SUHa for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation at week 3 after the start of treatment.

 twenty

Fig. 1F is a bar graph that represents the mean percentage (%) of reduction of plasma Ba levels (Y axis) over time in patients with SUHa (N = 35) after the start of treatment with eculizumab. The x-axis indicates the week of the visit of the patient with SUHa for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation at week 3 after the start of treatment. 25

Figs. 2A-2C are bar graphs that represent the percentage of patients with SUHa who achieve normalized concentrations of C5a in urine (Fig. 2A), sC5b9 in urine (Fig. 2B) and Ba in plasma (Fig. 2C) at baseline ( pretreatment with eculizumab) and several weeks after the start of treatment with eculizumab. 30

Fig. 3A is a dot plot depicting the concentration of prothrombin fragment 1 + 2 (in pmol / l) in the plasma of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after onset of treatment with eculizumab. The concentration of F1 + 2 in plasma was also measured in the plasma of normal, healthy (normal) individuals. 35

Fig. 3B is a dot diagram representing the concentration of D-dimer (in μg / l) in the plasma of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after the start of treatment with eculizumab. The concentration of D-dimer in plasma was also measured in the plasma of normal, healthy (normal) individuals. 40

Fig. 3C is a bar graph that represents the average percentage (%) of reduction of the levels of prothrombin fragments F1 + 2 in plasma (Y axis) over time in patients with SUHa after the start of treatment with eculizumab. The x-axis indicates the week of the visit of the patient with SUHa for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation in the 45 week 3 after the start of treatment.

Fig. 3D is a bar graph that represents the average percentage (%) of reduction of plasma d-dimer levels (Y axis) over time in patients with SUHa after the start of treatment with eculizumab. The x-axis indicates the week of the visit of the patient with SUHa for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation at week 3 after the start of treatment.

Figs. 4A and 4B are bar graphs that represent the percentage of patients with SUHa that achieve normalized concentrations of prothrombin fragment in plasma 1 + 2 (Fig. 4A) and plasma D-dimer (Fig. 55 4B) at baseline (pretreatment with eculizumab) and several weeks after the start of treatment with eculizumab.

Fig. 5A is a dot plot depicting the concentration of thrombomodulin (in ng / ml) in plasma (EDTA-treated plasma) of patients with SUHa both before treatment with eculizumab (Pre-Tx) 60 and several weeks later of the start of treatment with eculizumab. The plasma thrombomodulin concentration in the plasma of normal, healthy (normal) individuals was also measured. EOS designates the results of the analysis of samples obtained “at the end of the study”.

Fig. 5B is a dot plot representing the concentration of VCAM-1 (in ng / ml) in the serum of 65 patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after the start of treatment. with eculizumab. The serum VCAM-1 concentration in the serum of normal, healthy individuals (normal group) was also measured. EOS designates the results of the analysis of samples obtained “at the end of the study”.

Fig. 5C is a point diagram representing the activity of vWF (in mU / ml) in plasma (plasma 5 treated with EDTA) of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks later of the start of treatment with eculizumab. The activity of vWF in the plasma of normal, healthy (normal) individuals was also measured. EOS designates the results of the analysis of samples obtained “at the end of the study.

 10

Figs. 6A and 6B are bar graphs that represent the percentage of patients with SUHa that achieve normalized plasma thrombomodulin concentrations (Fig. 6A) and plasma vWF activity levels (Fig. 4B) at baseline (pretreatment with eculizumab) and several weeks after the start of treatment with eculizumab.

 fifteen

Fig. 6C is a bar graph that represents the average percentage (%) of reduction in plasma thrombomodulin levels (Y axis) over time in patients with SUHa (N = 33) after the start of treatment with eculizumab. The x-axis indicates the week of the visit of the patient with SUHa for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation at week 3 after the start of treatment. twenty

Fig. 6D is a bar graph that represents the mean percentage (%) of reduction in serum VCAM-1 levels (Y axis) over time in patients with SUHa (N = 36) after the onset of Eculizumab treatment. The x-axis indicates the week of the visit of the patient with SUHa for evaluation after the start of treatment, for example, V3 is the visit of the patient for evaluation at week 3 after the start of treatment.

Fig. 7A is a dot plot representing the concentration of TNFR1 (in pg / ml) in the serum of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after the start of treatment with eculizumab. The concentration of serum TNFR1 was also measured in the serum of 30 normal, healthy individuals (normal group). EOS designates the results of the analysis of samples obtained “at the end of the study”.

Fig. 7B is a bar graph that represents the percentage of patients with SUHa who achieve normalized serum TNFR1 concentrations at baseline (pretreatment with eculizumab) and several 35 weeks after the start of treatment with eculizumab.

Fig. 8A is a dot plot depicting the concentration of cystatin C (CysC) (in ng / mg creatine in urine) in the urine of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after the start of treatment with eculizumab. The concentration of 40 CysC in urine in the urine of normal, healthy individuals (NORM) was also measured.

Fig. 8B is a dot plot representing the concentration of β2M (in μg / mg of urine creatine) in the urine of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after the onset of Eculizumab treatment. The concentration of β2M in urine in the urine of normal, healthy individuals (NORM) was also measured.

Fig. 8C is a dot plot depicting the concentration of NGAL (in ng / mg of urine creatine) in the urine of patients with SUHa both before treatment with eculizumab (Pre-Tx) and several weeks after the onset of Eculizumab treatment. The concentration of NGAL in urine in the urine of normal, healthy individuals (NORM) was also measured.

Figs. 9A-9E are a series of bar graphs that represent the average levels of several SUHa biomarker proteins in patients with SUHa who underwent dialysis (dialysis), compared to patients with SUHa who did not undergo dialysis (without dialysis ) before inclusion in the study described in this document. Fig. 9A represents the mean serum TNFR1 concentration (in pg / ml); Fig. 9B represents the average concentration of β2M in urine (in μg / mg of creatine in urine); Fig. 9C represents the concentration of Ba in plasma (in ng / ml); Fig. 9D represents the concentration of sC5b9 in urine (in ng / mg of creatine in urine); and Fig. 9E represents the concentration of C5a in urine (in ng / ml).

 60

Fig. 10A is a dot plot representing the concentration of serum TNFR1 (in pg / ml) in patients with SUHa showing stable clinical parameters (clinical remission) (without TMA) and patients with SUHa who continue to experience levels of high haptoglobin and LDH (and reduced platelet counts) (others), both at baseline and at 1 to 2.5 weeks after the start of treatment with eculizumab. Serum TNFR1 concentrations of normal, healthy 65 (Normal) individuals are also shown.

Figs. 10B-10E are a series of bar graphs, each representing the concentration of a given biomarker in patients with normal hematological markers LDH and haptoglobin ("normal patients" or patients considered to be in clinical remission), patients with markers abnormal hematologic (elevated) ("abnormal patients" or patients with active SUHa presentation) and healthy ("normal") subjects. Fig. 10B represents plasma Ba levels (ng / ml) in these subject populations. Fig. 10C represents the level of serum VCAM-1 (ng / ml) in subject populations. Fig. 10E represents the level of prothrombin fragments 1 + 2 in plasma (pmol / l) in the populations and Fig. 10D represents the level of plasma D-dimer (in μg / l). The P values for the comparisons of respective groups are shown in the figures.

 10

Figs. 10F-10I are a series of bar graphs, each representing the concentration of a given biomarker in patients with normal platelet levels ("normal patients"), patients with abnormal platelet levels (reduced) (abnormal patients "), and subjects healthy ("normal"). Fig. 10F represents plasma Ba levels (ng / ml) in these subject populations. Fig. 10G represents the level of serum VCAM-1 (ng / ml) in subject populations. Fig. 10I represents the level of prothrombin fragments 1 + 2 15 in plasma (pmol / l) in the populations and Fig. 10H represents the level of plasma D-dimer (in μg / l). The P values for the comparisons of respective groups are shown in the figures.

Fig. 11 is a bar graph depicting the mean percentage of change in serum and clusterin TNFR1 levels, C5a and C5b9 in urine in patients with SUHa who achieve a complete TMA 20 response and patients still experiencing TMA events (incomplete response). (As noted and elaborated in the working examples, a complete TMA response refers to a normalization of hematological parameters and preservation of renal function.)

Fig. 12 is a bar graph that represents the average percentage of change in plasma levels of Ba in patients with SUHa treated with eculizumab who experience a complete TMA response and patients with SUHa treated with eculizumab who do not (others ).

Fig. 13 is a bar graph depicting the mean change from baseline (initial visit, before treatment with eculizumab) in platelet count (109 / l) at weeks 12-17 and week 26 after 30 initiation of treatment with eculizumab in patients with SUHa with normalized plasma Ba levels versus persistently elevated plasma BA levels. The p values for each observation are also provided in the figure.

Figs. 14A-14D are a series of bar graphs that represent the observation that certain 35 biomarkers associated with SUHa are elevated in patients with SUHa with abnormal baseline TMA markers. Fig. 14A represents the concentration of cystatin C (CysC) (in ng / mg of urine creatine) in the urine of patients with SUHa with normal platelet counts (> 150,000 per μl of blood) compared to patients with reduced platelet counts (<150,000 per μl of blood). Fig. 14B represents the concentration of clusterin (in ng / mg of urine creatine) in the urine of patients with SUHa with 40 normal platelet counts (> 150,000 per μl of blood) compared to patients with reduced platelet counts (<150,000 per μl of blood). Fig. 14C represents the concentration of VCAM-1 in the serum of patients with SUHa with normal LDH levels compared to patients with elevated LDH levels. Fig. 14D represents the concentration of d-dimer (in μg / l) in the plasma of patients with SUHa with normal LDH levels compared to patients with elevated LDH levels. The p values for each observation are indicated in the figures.

Fig. 15 is a bar graph depicting the level of cystatin C (ng / mg creatine in urine) in the urine of patients with baseline SUHa who have repeated plasma therapy (repeated PT; N = 23), no plasma therapy (no PT; N = 3) or in normal patients (N = 9). fifty

Fig. 16 is a series of bar graphs representing the mean change of baseline eGFR (ml / min / 1.73 m2) in patients with SUHa who achieve normalized levels of various biomarkers (Ba in plasma, VCAM-1 in serum, F1 + 2 in plasma, d-dimer in plasma and cystatin C in urine) after treatment with eculizumab compared to patients with SUHa in which the concentration of these biomarkers 55 remains high.

Figs. 17A-E are a series of bar graphs that represent the observation that certain biomarkers associated with SUHa are elevated in patients with SUHa before treatment with a complement inhibitor, regardless of whether patients have received 60 plasma exchange therapy ( PE) or plasma infusion (PI). Fig. 17A depicts the concentration of the proteolytic fragment of factor B Ba (in ng / ml) in the plasma of normal healthy volunteers (VSN), patients with SUHa receiving PE or PI therapy (PE / PI) or patients with SUHa who do not receive PE / PI therapy (without PE / PI). Fig. 17B represents the concentration of sTNFR1 (in pg / ml) in the serum of normal healthy volunteers (VSN), patients with SUHa receiving PE or PI therapy (PE / PI), or patients with SUHa who do not receive therapy of PE / PI (no 65 PE / PI). Fig. 17C represents the concentration of sVCAM-1 (in ng / ml) in the serum of normal healthy volunteers (VSN), patients with SUHa receiving PE or PI therapy (PE / PI) or patients with SUHa who do not receive PE / PI therapy (without PE / PI). Fig. 17D represents the concentration of D-dimer (in μg / L) in the plasma of normal healthy volunteers (VSN), patients with SUHa receiving PE or PI therapy (PE / PI) or patients with SUHa who do not receive PE / PI therapy (without PE / PI). Fig. 17E represents the concentration of cystatin-C (in ng / mg of creatinine in urine) in the urine of normal healthy volunteers (VSN), patients with SUHa 5 receiving PE or PI therapy (PE / PI) or patients with SUHa who do not receive PE / PI therapy (without PE / PI). The p values for each observation are indicated in the figures.

Figs. 18A-E are a series of bar graphs that represent the observation that certain biomarkers associated with SUHa are elevated in patients with SUHa before treatment with a complement inhibitor, regardless of platelet levels in patients. Fig. 18A represents the concentration of the proteolytic fragment of factor B Ba (in ng / ml) in the plasma of normal healthy volunteers (VSN), patients with SUHa who have normal platelet levels (> 150 x 109) or patients with SUHa who they have reduced platelet counts (<150 x 109). Fig. 18B represents the concentration of sTNFR1 (in pg / ml) in the serum of normal healthy volunteers (VSN), patients with SUHa who have normal platelet levels (> 150 x 109) or patients with SUHa who have reduced platelet counts (<150 x 109). Fig. 18C represents the concentration of sVCAM-1 (in ng / ml) in the serum of normal healthy volunteers (VSN), patients with SUHa who have normal platelet levels (> 150 x 109) or patients with SUHa who have platelet counts reduced (<150 x 109). Fig. 18D represents the concentration of D-dimer (in μg / l) in the plasma of normal healthy volunteers (VSN), patients with SUHa who have normal platelet levels (> 150 x 109) or patients with SUHa who have counts reduced platelets (<150 x 109). Fig. 18E represents the concentration of cystatin-C (in ng / mg of creatinine in urine) in the urine of normal healthy volunteers (VSN), patients with SUHa who have normal platelet levels (> 150 x 109) or patients with SUHa that have reduced platelet counts (<150 x 109). The p values for each observation are indicated in the figures. 25

Figs. 19A-E are a series of bar graphs that represent the observation that certain biomarkers associated with SUHa are elevated in patients with SUHa before treatment with a complement inhibitor, regardless of levels of haptoglobin (Hp) or lactate dehydrogenase (LDH ). Fig. 19A depicts the concentration of the proteolytic fragment of factor B Ba (in ng / ml) in plasma 30 of normal healthy volunteers (VSN), patients with SUHa who have normal Hp and LDH levels, or patients with SUHa who have Hp / LDH elevated (abnormal). Fig. 19B represents the concentration of sTNFR1 (in pg / ml) in the serum of normal healthy volunteers (VSN), patients with SUHa that have normal Hp and LDH levels, or patients with SUHa who have elevated Hp / LDH (abnormal ). Fig. 19C represents the concentration of sVCAM-1 (in ng / ml) in the serum of normal healthy volunteers (VSN), patients with SUHa 35 who have normal Hp and LDH levels, or patients with SUHa who have Hp / LDH elevated (anomalous). Fig. 19D represents the concentration of D-dimer (in μg / l) in the plasma of normal healthy volunteers (VSN), patients with SUHa that have normal Hp and LDH levels, or patients with SUHa who have elevated Hp / LDH (anomalous). Fig. 19E represents the concentration of cystatin-C (in ng / mg of creatinine in urine) in the urine of normal healthy volunteers (VSN), patients with SUHa who have normal Hp and LDH levels, or 40 patients with SUHa who they have elevated Hp / LDH (abnormal). The p values for each observation are indicated in the figures.

Figs. 20A-B are box-whisker diagrams that represent the longitudinal effects of sustained eculizumab treatment on terminal complement activation in patients with SUHa. Fig. 20A represents the change over time in the concentration of urine C5a (ng / mg creatinine in urine) of patients with SUHa after treatment with eculizumab, compared with the concentration of C5a in urine in the urine. urine of normal healthy volunteers (VSN). Fig. 20B represents the change over time in the concentration of sC5b-9 in urine (ng / mg creatinine in urine) of patients with SUHa after treatment with eculizumab, compared to the concentration of sC5b-9 in urine in the urine of normal healthy volunteers 50 (VSN). The box-mustache diagrams show the median, the 25th and 75th percentiles and the interval. * The first time point at which levels were significantly reduced against the baseline (LB); P values against the baseline at each time point were calculated using a repeated measures approach based on maximum restricted probability (mixed model). P values compared to VSN were calculated using the Wilcoxon range sum test. 55

Figs. 21A-C are box-whisker diagrams that represent the longitudinal effects of sustained eculizumab treatment in the concentration of biomarker proteins associated with renal injury in patients with SUHa. Fig. 20A represents the change over time in the concentration of urine FABP-1 (ng / mg creatinine in urine) of patients with SUHa after treatment with eculizumab, compared with the concentration of FABP-1 in urine in the urine of normal healthy volunteers (VSN). Fig. 21B represents the change over time in the concentration of cystatin C in urine (ng / mg creatinine in urine) of patients with SUHa after treatment with eculizumab, compared to the concentration of cystatin C in urine in the urine of normal healthy volunteers (VSN). Fig. 21C represents the change over time in the concentration of urine clusterin (ng / mg creatinine in urine) of patients with SUHa 65 after treatment with eculizumab, compared to the concentration of urine clusterin in the urine of normal healthy volunteers (VSN). The box-mustache diagrams show the median, the 25th and 75th percentiles and the interval. * The first time point at which levels were significantly reduced against the baseline (LB); P values against the baseline at each time point were calculated using a repeated measures approach based on maximum restricted probability (mixed model). P values compared to VSN were calculated using the Wilcoxon range sum test. 5

Fig. 22 is a box-mustache diagram depicting the longitudinal effects of sustained eculizumab treatment in the activation of the alternative complement pathway in patients with SUHa. The change over time in the concentration of Ba (ng / ml) in the plasma of patients with SUHa after treatment with eculizumab is shown together with the concentration of Ba in plasma in normal healthy volunteers (VSN). The box-mustache diagram shows the median, the 25th and 75th percentiles and the interval. * The first time point at which levels were significantly reduced against the baseline (LB); P values versus baseline at each time point were calculated using a repeated measures approach based on maximum restricted probability (mixed model). P values compared to VSN were calculated using the Wilcoxon range sum test. fifteen

Figs. 23A-C are box-whisker diagrams that represent the longitudinal effects of sustained eculizumab treatment in the concentration of biomarker proteins associated with inflammation, activation of endothelial cells and tissue damage in patients with SUHa. Fig. 23A represents the change over time in the concentration of sTNFR1 (pg / ml) in the serum of patients with SUHa after treatment with eculizumab, compared to the concentration of sTNFR1 in the serum of normal healthy volunteers. (VSN). Fig. 23B represents the change over time in the concentration of sVCAM-1 (ng / ml) in the serum of patients with SUHa after treatment with eculizumab, compared with the concentration of analyte in the serum of healthy volunteers. normal (VSN). Fig. 23C represents the change over time in the concentration of thrombomodulin (ng / ml) in the plasma of patients with SUHa after treatment with eculizumab, compared with the concentration of the analyte in the plasma of normal healthy volunteers (VSN). The box-mustache diagrams show the median, 25th and 75th percentiles and the interval. * The first time point at which levels were significantly reduced against the baseline (LB); P values versus baseline at each time point were calculated using a repeated measures approach based on maximum restricted probability (mixed model). The P values compared to VSN were calculated using the Wilcoxon ranks sum 30 test.

Figs. 24A-B are box-whisker diagrams that represent the longitudinal effects of sustained eculizumab treatment in the concentration of biomarker proteins associated with thrombosis and coagulation in patients with SUHa. Fig. 24A represents the change over time in the concentration of F1 + 2 35 (pmol / l) in the plasma of patients with SUHa after treatment with eculizumab, compared to the analyte concentration in the plasma of volunteers normal healthy (VSN). Fig. 24B represents the change over time in the concentration of D-dimer (mg / l) in the plasma of patients with SUHa after treatment with eculizumab, compared with the concentration of analyte in the plasma of healthy volunteers normal (VSN). The box-mustache diagrams show the median, 25th and 75th percentiles and the interval. * The first time point at which levels were significantly reduced against the baseline (LB); P values against the baseline at each time point were calculated using a repeated measures approach based on maximum restricted probability (mixed model). P values compared to VSN were calculated using the Wilcoxon range sum test.

 Four. Five

Overview of the complement system

The complement system acts together with other immune systems of the body to defend against the intrusion of cellular and viral pathogens. There are at least 25 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors. Plasma proteins make up approximately 10% of globulins in vertebrate serum. Complement components achieve their immune defensive functions by interacting in a series of intricate but precise membrane binding and enzymatic cleavage events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory and lytic functions. A concise summary of the biological activities associated with complement activation is provided, for example, in The Merck Manual, 16th edition.

The complement cascade progresses through the classical route, the alternative route or the lectin route. These routes share many components, and although they differ in their initial stages, they converge and share the same "terminal complement" components (C5 to C9) responsible for the activation and destruction of target cells. 60

The classical route (RC) is typically initiated by the recognition by antibodies of, and binding to, an antigenic site in a target cell. The alternative route (RA) may be antibody independent, and may be initiated by certain molecules on pathogen surfaces. Additionally, the lectin pathway typically begins with the binding of mannose binding lectin (LUM) with high mannose substrates. These routes converge at the point where the component of complement C3 is cleaved by an active protease to produce C3a and C3b. Other routes that activate the complement attack may subsequently act in the sequence of events that lead to various aspects of the complement function. C3a is an anaphylatoxin. C3b binds with bacterial and other cells, as well as with certain viruses and immune complexes, and marks them for withdrawal from circulation. This opsonic function of C3b is generally considered the most important anti-infective action of the complement system. C3b also forms a complex with other unique components of each route to form classic or alternative C5 convertase, which cleaves the complement component C5 (hereafter referred to herein as "C5") into C5a and C5b.

The cleavage of C5 releases biologically active species such as C5a, a potent anaphylatoxin and chemotactic factor and C5b that through a series of protein interactions leads to the formation of the complex of the lithic terminal complement, C5b-9. C5a and C5b-9 also have activating properties of pleiotropic cells, amplifying the release of inflammatory factors downstream, such as hydrolytic enzymes, reactive oxygen species, arachidonic acid metabolites and various cytokines.

C5b combines with C6, C7 and C8 to form the complex of C5b-8 on the surface of the target cell. Following the binding of several C9 molecules, the membrane attack complex (CAM, C5b-9, terminal complement complex-CCT) is formed. When sufficient numbers of CAMs are inserted into target cell membranes, the openings they create (CAM pores) mediate the rapid osmotic lysis of the target cells. Smaller, non-lytic concentrations of CAM may produce other effects. In particular, membrane insertion of small numbers of C5b-9 complexes into endothelial cells and platelets can cause deleterious cell activation. In some cases, activation may precede cell lysis.

As mentioned above, C3a and C5a are activated complement components. These can trigger mast cell degranulation, which releases histamine from basophils and mast cells, and other mediators of inflammation, resulting in smooth muscle contraction, increased vascular permeability, leukocyte activation and other inflammatory phenomena including cell proliferation that results in Hypercellularity result. C5a also acts as a chemotactic peptide that acts to attract proinflammatory granulocytes to the complement activation site. C5a receptors are found on the surfaces of bronchial and alveolar epithelial cells and bronchial smooth muscle cells. C5a receptors have also been discovered in eosinophils, mast cells, monocytes, neutrophils and activated lymphocytes. 30

Detailed description

As described herein and exemplified in the working examples, the inventors have identified biomarkers for SUHa. For example, it has been found that a high or, in some cases, reduced concentration of certain proteins is associated with the presence of SUHa. Similarly, a high or reduced concentration (or activity) of certain proteins in a biological liquid obtained from a patient with SUHa treated with a complement inhibitor indicates that the patient has responded to therapy with the inhibitor. Consequently, the analysis of the concentration and / or the level of activity of said proteins can be used to evaluate, among other things, the risk of SUHa, diagnosis of SUHa, supervision of the progression or reduction of SUHa, and / or supervision of the response to treatment to a complement inhibitor.

SUHa biomarker proteins and applications

SUHa biomarker proteins are exposed (as well as exemplary biological fluids in which they are found) 45 in Table 1. The protein sequence associated with the name of each of the biomarkers listed in Table 1 in GenBank (National Center for Information Biotechnology (NCBI)) is available on the date of submission of this application.

Table 1. 50

 Biomarker

 Apr. Tissue source Seq no. NCBI reference *

 Serum

 Urine Plasma

 Markers of inflammation / activation of platelets or endothelial

 Chemokine Ligand (motif C-X-C) 9

 CXCL9 X NP_002407.1

 Chemokine Ligand (motif C-X-C) 10

 CXCL-10 X NP_001556.2

 Interleukin-1 beta

 IL-1β X NP_000567.1

 Interleukin-6

 IL-6 X NP_000591.1

 Interleukin-8

 IL-8 X NP_000575.1

 Interleukin-12 p70

 IL-12p70 X NP_000873.2 (p35) NP_002178.2 (p40)

 Interferon-gamma

 IFN-γ X NP_000610.2

 Platelet Selectin

 p-selectin X NP_002996.2

 Biomarker

 Apr. Tissue source Seq no. NCBI reference *

 Biomarker

 Apr. Tissue source Seq no. NCBI reference *

 Serum

 Urine Plasma

 Endothelial selectin

 e-selectin X NP_000441.2

 Intercellular adhesion molecule 1

 ICAM-1 X NP_000192.2

 Adhesion molecule of vascular cells 1

 VCAM-1 X NP_001069.1

 Monocyte chemotactic protein 1

 MCP-1 X NP_002973.1

 Vascular Endothelial Growth Factor

 VEGF X NP_001020537.2

 Regulated in activation, expressed in normal T cells and secreted (CCL5)

 CCL5 X NP_002976.2

 Soluble CD40 Ligand

 sCD40L X NP_000065.1 **

 Soluble tumor necrosis factor receptor 1

 sTNFR1 X NP_001056.1 **

 Interleukin-18

 IL-18 X NP_001553.1

 Inflammation / kidney injury markers

 Lipocalin associated with neutrophil gelatinase

 NGAL X NP_005555.2

 1 kidney injury molecule

 KIM-1 X NP_001092884.1

 Osteopontin

 OPN X NP_001035147.1

 Tissue inhibitor of metalloproteinases 1

 TIMP-1 X NP_003245.1

 Interleukin-18

 IL-18 X Mentioned above

 Chemokine Ligand (motif C-X-C) 9

 CXCL9 X Mentioned above

 Chemokine Ligand (motif C-X-C) 10

 CXCL10 X Mentioned above

 Clusterin

 CLU X NP_001822.3

 Cystatin C

 CyC X NP_000090.1

 Albumin

 ALB X NP_000468.1

 Acid binding protein of liver grades

 L-FABP X NP_001434.1

 Beta 2 microglobulin

 β2M X NP_004039.1

 Clover Factor 3

 TFF-3 X NP_003217.3

 N-acetyl-beta-D-glucosaminidase

 NAG X NP_000511.2

 π-glutathione S-transferase

 % -GST X NP_000843.1

 Alpha-glutathione S-transferase

 α-GST X NP_665683.1

 Complement

 Ba complement

 Ba X SEQ ID NO: 1; See, also Fig. 2 of Morley and Campbell (1984) EMBO J 3 (1): 153-157.

 C3a complement

 C3a X SEQ ID NO: 2

 C5a complement

 C5a X X SEQ ID NO: 3

 Soluble CAM

 sC5b9 X X ND

 CH50 (hemolysis)

 CH50 X ND

 C5 complement

 C5 X X NP_001726.2

 Thrombosis / coagulation

 D-dimer

 D-dimer

    X P02671 ***

 Prothrombin F1 + 2

 F1 + 2 X Activation fragment 1 (SEQ ID NO: 4) corresponds to amino acids 44-198 of SEQ ID NO: 6. Activation fragment 2 (SEQ ID NO: 5) corresponds to amino acids 199-327 of SEQ ID NO: 4.

 Von Willebrand factor

 vWF X NP_000543.2

 Von Willebrand factor activity

 vWF X activity Same reference

 Thrombomodulin

 TM X NP_000352.1

 Serum

 Urine Plasma

 * The NCBI reference number for an exemplary human sequence is provided for each biomarker protein listed in the Table. ** The soluble form of the receptor is generated by proteolytic processing of the membrane-bound form of the receptor. *** Designation UniProtKB (consortium: European Bioinformatics Institute, Cambridge, United Kingdom; Swiss Institute of Bioinformatics; Geneva, Switzerland; and Protein Information Service, Washington, DC) for human alpha fibrinogen, which is cleaved by thrombin to form fibrin. D-dimer is a fibrin degradation product. A description of the transition based on cleavage of fibrinogen to fibrin to D-dimer is set forth in Soheir et al. (2009) Blood 113 (13): 2878-2887.

The biomarkers provided herein may be used alone or in combination as an indicator to, for example, assess the risk of developing SUHa, diagnose SUHa, determine if a subject is experiencing the first acute presentation of SUHa, monitor progression or reduction. of SUHa, and / or monitor the response to treatment with a complement inhibitor or optimize said treatment. In some embodiments, an individual SUHa biomarker protein described herein can be used. In some embodiments, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, may be used. 23, 24 or 25 or more) SUHa biomarker proteins of Table 1 in combination as a panel.

 10

In some embodiments, SUHa biomarker proteins are selected from a proteolytic fragment of complement component component B (eg, Ba or Bb), soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, sTNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, β2 microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-15 1, IL-18, vascular endothelial cell growth factor ( VEGF), IL-6, albumin, IL-8 and include sTNFR1. The concentration and / or activity of one or more of the biomarkers can be measured in Table 1 (or any of the subsets of biomarkers mentioned herein).

In some embodiments, an increase in the d-dimer concentration, relative to the d-20 dimer concentration in a normal control sample, and an increase in the concentration of FABP-1, relative to the concentration of FABP- 1 in a normal control sample, indicates that the patient with SUHa experiences a first manifestation of acute SUHa. In some cases, that increase in one or both of these SUHa biomarker proteins is a significant increase compared to normal control.

 25

In some embodiments, an increase in the concentration of one or more of sTNFR1, Ba, C5b-9, F1 + 2, β2M, clusterin, TIMP-1, NGAL, CysC and C5a (see Table 7) in a biological sample obtained from a patient with SUHa, in relation to the control concentration of the analytes obtained, for example, from a group of samples of patients with SUHa who have not received repeated dialysis, indicates that the patient is one who has received repeated dialysis. 30

In some embodiments, an increase in the concentration of one or both of C5a and FABP-1 (for example, C5a and FABP-1 in urine) in a biological sample obtained from a patient with SUHa, relative to the control concentration of Analytes obtained, for example, from a group of samples of patients with SUHa who have not received a kidney transplant, indicates that the patient is one who has received a kidney transplant. 35

In some embodiments, an increase in the concentration of cystatin C (for example, cystatin C in urine) in a biological sample obtained from a patient with SUHa, relative to the control concentration of the analytes obtained, for example, from a group of samples of patients with SUHa who have not received repeated plasma therapy, indicates that the patient is one who has received repeated plasma therapy. 40

In some embodiments, a reduction after treatment of the concentration of Ba (for example, concentration of Ba in plasma) of at least 10 (for example, at least 15, 20, 25, 30, 35, 40, 45 or 50) %, in relation to the concentration of Ba in a sample of the same type of biological fluid obtained from the subject before treatment, indicates that the subject has or is likely to achieve a complete thrombomicroangiopathy (TMA) response (i.e. cessation of TMA events). In some embodiments, the reduction occurs at week 12 after the first treatment with the complement inhibitor. In some embodiments, the reduction occurs in weeks 12-17 after the first treatment with the complement inhibitor. In some embodiments, the reduction occurs at week 26 after the first treatment with the complement inhibitor. fifty

In some embodiments, a reduction after treatment in one or both of CCL5 and sCD40L of at least 10 (for example, at least 15, 20, 25, 30, 35, 40, 45 or 50)%, relative to the concentration In a sample or samples of the same type of biological fluid obtained from the subject before treatment, it indicates that the subject has or is likely to get increased platelet counts (for example, platelet recovery). In 55

some embodiments, a reduction after treatment of the concentration of Ba (for example, concentration of Ba in plasma) of at least 10 (for example, at least 15, 20, 25, 30, 35, 40, 45 or 50)% (or normalization of Ba concentrations), in relation to the concentration of Ba in a sample of the same type of biological fluid obtained from the subject before treatment, indicates that the subject has or is likely to have achieved a thrombomicroangiopathy (TMA) response ) complete (i.e. cessation of TMA events). 5

In some embodiments, the status of one or more of the SUHa biomarkers described herein may be predictive of improvement in the estimated glomerular filtration rate (eGFR) for a patient with SUHa treated with a complement inhibitor. For example, a reduction in the concentration of prothrombin F1 + 2 (for example, in 4, 5 or 6 weeks after initial treatment in a chronic treatment regimen) and / or d-10 dimer (for example, in 12, 13 , 14, 15, 16 or 17 weeks after initial treatment in a chronic treatment regimen) indicates that a patient with SUHa treated with a complement inhibitor has achieved or is likely to achieve a clinically significant improvement in eGFR. The attainment or probable achievement of a clinically significant improvement in eGFR is also indicated by a normalization of the concentration of IL-6 and IFN-γ (for example, in 4, 5 or 6 weeks after initial treatment with a complement inhibitor in 15 a chronic treatment regimen). The achievement or probable achievement of a clinically significant improvement of eGFR is also indicated by a normalization of the concentration of Ba, CXCL9, CXCL10 and vWF (for example, in 12, 13, 14, 15, 16 or 17 weeks after the initial treatment with a complement inhibitor in a chronic treatment regimen). In some embodiments, the achievement or probable achievement of a clinically significant improvement of eGFR is also indicated by a normalization of the concentration of Ba, CXCL9, CXCL10 β2M (for example, in urine), CysC (for example, in urine), vWF, d-dimer, clusterin (for example, in urine) and / or FABP-1 (for example, in urine) (for example, in 26 weeks after initial treatment with a complement inhibitor in a chronic treatment regimen) .

Methods for monitoring or evaluating the status of one or more biomarker proteins associated with atypical hemolytic uremic syndrome (SUHa) in a subject (eg, a mammal, for example, a human being) include: measuring in a biological liquid obtained from the subject one or both of (i) the concentration of at least one (for example, at least two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) biomarker protein associated with SUHa in the biological liquid.

 30

Measurement or determination of protein expression levels in a biological sample can be performed by any suitable method (see, for example, Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory: Cold Spring Harbor, NY ). In general, protein levels are determined by contacting a biological sample obtained from a subject with binding agents for one or more of the SUHa biomarker proteins; detecting, in the sample (for example, the biological liquid), the levels of one or more of the SUHa biomarker proteins that bind with the binding agents; and comparing the levels of one or more of the SUHa biomarker proteins in the sample with the corresponding protein biomarker levels in a control sample (eg, a normal sample). In certain embodiments, a suitable binding agent is a ribosome, with or without a peptide component, an RNA molecule or a polypeptide (for example, a polypeptide comprising a polypeptide sequence of a protein marker, a peptide variant 40 thereof or a non-peptide mimetic of said sequence.

Suitable binding agents also include an antibody specific for a SUHa biomarker protein described herein (for example, an antibody specific for any biomarker listed in Table 1). Antibodies suitable for use in the methods of the present invention include monoclonal and polyclonal antibodies and antigen-binding fragments (eg, Fab fragments or scFvs) of antibodies. Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, can be prepared using methods known in the art (see, for example, Kohler and Milstein (1975) Nature 256: 495-497; Kozbor et al. (1985) J Immunol Methods 81 : 31-42; Cote et al. (1983) Proc Natl Acad Sci USA 80: 2026-203; and Zhang et al. (2002) JBiol Chem 277: 39379-39387). Antibodies for use in the methods of the invention can be purified by methods well known in the art. Antibodies can also be obtained from commercial sources.

In certain embodiments, the binding agent is directly or indirectly labeled with a detectable moiety. The role of a detectable agent is to facilitate the detection step of the diagnostic method by allowing the visualization of the complex formed by the union of the binding agent with the protein marker (or fragment thereof). The detectable agent can be selected so that it generates a signal that can be measured and whose intensity is related (preferably proportional) to the amount of protein marker present in the sample being analyzed. Methods for labeling biological molecules such as polypeptides and antibodies are well known in the art. Any of a wide variety of detectable agents can be used in the practice of the present invention. Suitable detectable agents include, but are not limited to: various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used in an ELISA, that is, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric markers, magnetic markers and biotin, digoxigenin or other haptens and proteins for which antisera or 65 monoclonal antibodies are available.

In certain embodiments, the binding agents (eg, antibodies) can be immobilized in a carrier or carrier (for example, a bead, a magnetic particle, a latex particle, a microtiter plate well, a cuvette or other vessel of reaction). Examples of suitable carrier or carrier materials include agarose, cellulose, nitrocellulose, dextran, Sephadex®, Sepharose®, liposomes, carboxymethylcellulose, polyacrylamides, polystyrene, gabbros, filter paper, magnetite, ion exchange resin, plastic film, tube plastic, 5 glass, polyamine methyl vinyl ether maleic acid copolymer, amino acid copolymer, ethylene maleic acid copolymer, nylon, silk and the like. Binding agents can be immobilized indirectly using specific secondary binding agents for the first binding agents (for example, mouse antibodies specific for protein markers can be immobilized using specific sheep antibody anti mouse Fc IgG fragment applied on the vehicle or support). 10

Protein expression levels in a biological sample can be determined using immunoassays. Examples of such assays are time-resolved fluorescence immunoassays (TR-FIA), radioimmunoassays, enzyme immunoassays (eg, ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, Western blotting and histochemical assays, which they are conventional methods well known in the art. The methods of detecting and quantifying the signal generated by the complex formed by binding the binding agent with the protein marker will depend on the nature of the assay and the detectable moiety (eg, fluorescent moiety).

In one example, the presence or amount of protein expression of a gene (for example, a biomarker protein of 20 SUHa depicted in Table 1) can be determined using a Western blotting technique. For example, a lysate can be prepared from a biological sample, or the biological sample (for example, biological liquid) itself, can be contacted with Laemmli buffer and electrophoresed in sodium dodecyl sulfate polyacrylamide gel ( SDS-PAGE). Proteins resolved by SDS-PAGE, separated by size, can be transferred to a filter membrane (eg, nitrocellulose) and subjected to immunoblot techniques using a detectably labeled antibody specific for the protein of interest. The presence or amount of detectably labeled antibody bound indicates the presence or amount of protein in the biological sample.

In another example, an immunoassay can be used to detect and / or measure the protein expression of a SUHa biomarker protein (for example, one represented in Table 1). As before, for detection purposes, an immunoassay can be performed with an antibody carrying a detection moiety (for example, a fluorescent agent or an enzyme). Proteins from a biological sample can be conjugated directly with a solid phase matrix (for example, a multiwell test plate, nitrocellulose, agarose, Sepharose®, coded particles or magnetic beads) or it can be conjugated with a first member of a specific binding pair (for example, biotin or streptavidin) that binds with a solid phase matrix after binding with a second member of the specific binding pair (eg, streptavidin or biotin). Such binding with a solid phase matrix allows the proteins to be removed by purification of other interfering or irrelevant components of the biological sample before contact with the detection antibody and also allows subsequent washing of unbound antibody. Here, as before, the presence or amount of detectably labeled antibody bound indicates the presence or amount of protein in the biological sample.

Alternatively, protein expression levels can be determined using mass spectrometry based methods or image capture based methods known in the art for protein detection. Other suitable methods include two-dimensional gel electrophoresis, proteomic-based methods, such as the identification of individual proteins recovered from the gel (eg, by mass spectrometry or N-terminal sequencing) and / or bioinformatics.

Optionally, methods can be performed to detect or measure protein expression in formats that allow rapid preparation, processing and analysis of multiple samples. This may be, for example, in multiwell test plates (for example, 96 wells or 386 wells) or matrices (for example, protein microplates). Stock solutions for various reagents can be provided manually or robotically, and sample preparation, pipetting, dilution, mixing, distribution, washing, incubation (eg, hybridization), sample reading, data collection (optical data) and / can be performed. or subsequent analysis (computer-assisted image analysis) in a robotic way using analysis, robotics and detection instrumentation software available in the market capable of detecting the signal generated from the test. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters and devices that measure radioisotopic degradation. Exemplary high-performance cell-based assays (for example, detecting the presence or level of a target protein in a cell) can use the HCS ArrayScan® VTI Reader or KineticScan® HCS Reader Technology (Cellomics Inc., Pittsburg, PA). 60

Methods for determining the activity of vWF are also known in the art and are described herein (eg, working examples). See also, for example, Horvath et al. (2004) Exp Clin Cardiol 9 (10): 31-34. Commercial kits, Instrumentation Laboratory (Bedford, MA; catalog number: 0020004700) and Quest Diagnostics (Madison, NJ) are also available. 65

In some embodiments, the level of expression (or activity) of proteins of at least two SUHa biomarker proteins (for example, at least three proteins, at least four proteins, at least five proteins, at least six proteins, can be evaluated and / or measured) proteins, at least seven proteins, at least eight proteins, at least nine proteins, at least 10 proteins, at least 11 proteins, at least 12 proteins, at least 13 proteins, at least 14 proteins, at least 15 proteins, at least 16 proteins, at least 17 proteins, at least 18 proteins, at least 19 proteins, at least 20 proteins, at least 21 proteins, at least 22 proteins, at least 23 proteins, or at least 24 proteins or more).

In some embodiments, the biological fluid in which SUHa biomarker proteins are measured is blood. In some embodiments, the biological liquid is a fraction of blood, for example, serum or plasma. In some 10 embodiments, the biological liquid is urine. In some embodiments, all measurements are performed on a biological liquid sample (for example, a serum sample). In some embodiments, measurements are made in at least two different biological fluids obtained from the subject. For example, in some embodiments, the concentration or activity of one or more SUHa biomarker proteins is measured in a serum sample obtained from the patient. In some embodiments, a blood sample and a urine sample 15 are available to allow testing of different analytes in two different sample matrices.

The subject may be, for example, a human being who has, is suspected of having or is at risk of developing SUHa. The subject may be one that has been treated (or is being treated) with a complement inhibitor (eg, an inhibitor of complement component C5 such as an anti C5 antibody). The treatment may have occurred less than one month (for example, less than 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days) before obtaining the sample of the subject.

The method may also include the step of determining if the subject has or is at risk of developing SUHa. When the subject has been treated or is being treated with a complement inhibitor (for example, an anti-C5 antibody) in a predetermined dosage schedule, the method may further include determining whether the patient is (therapeutically) sensitive to the inhibitory therapy. of the complement.

In some embodiments of any of the methods described herein, the method requires recording the measured value or values of the concentration of the at least one SUHa biomarker protein. The record can be written or in a computer readable medium. The method may also include communicating the measured value or values of the concentration of the at least one SUHa biomarker protein to the subject and / or a medical practitioner in whose charge the subject has been placed.

In some embodiments, any of the methods described herein may include the step of administering to the subject the complement inhibitor at a higher dose or with an increased dosage frequency, in relation to the predetermined dosage schedule, if the subject It is not sensitive to treatment with the inhibitor in the predetermined dosage schedule.

Some of the methods described herein involve comparing the measured concentration or activity of a SUHa biomarker protein (as measured in a biological sample obtained from a subject) with a control sample. In some embodiments, control sample of the subject is obtained before administering to the subject a complement inhibitor (for example, a C5 inhibitor such as eculizumab). In some embodiments, the control sample may be (or may be based on), for example, a collection of samples obtained from one or more (eg, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35 or 40 or more) healthy individuals to whom a complement inhibitor has not been administered. In some embodiments, the control sample may be (or may be based on), for example, a pooled sample obtained from two or more (eg, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35 or 40 or more) individuals. In some embodiments of any of the methods described herein, the pooled samples may be from healthy individuals or, at least, individuals who do not have or are not suspected of having (are not at risk of developing) SUHa. For example, determining whether a subject is one who has SUHa may involve comparing the measured concentration of one or more serum biomarkers in the subject and comparing the measured concentration with the average concentration of the same biomarkers in the pooled healthy samples. Similarly, determining whether the concentration or activity of a biomarker associated with SUHa has been reduced after treatment with a complement inhibitor may involve comparing the concentration or activity of the protein in a biological liquid obtained from a subject before treatment with a complement inhibitor with the protein concentration in a sample of the same biological liquid obtained from the patient after treatment with the inhibitor (for example, one day, two days, three days, four days, five days, six days, 1 week, 2 weeks, 3 weeks, one month, 6 weeks, two months or three months after treatment (for example, the first of a series of treatments in chronic therapy) with the inhibitor). 60

In some embodiments, the determination of whether a complement inhibitor has produced a desired effect (for example, a reduction in the concentration or activity of a SUHa biomarker protein) in a human being can be done by investigating whether the post-treatment concentration of the protein remains. within a predetermined range indicative of sensitivity to a complement inhibitor by a human being. In some embodiments, the determination of whether a complement inhibitor has produced a desired effect on a being

Human may include investigating whether the post-treatment concentration or activity of one or more SUHa biomarker proteins is above or below a predetermined cut-off value. A cut-off value is typically the concentration or activity of a given protein in a given biological liquid above or below what is considered indicative of a certain phenotype, for example, sensitivity to therapy with a complement inhibitor. 5

In some embodiments of any of the methods described herein, the same practitioner may administer the complement inhibitor to the subject before determining whether there has been a change in the concentration or activity of one or more SUHa biomarker proteins, while In some embodiments, the practitioner who administers the inhibitor to the subject is different from the practitioner who determines whether a response has occurred in the subject. In some embodiments, the practitioner may obtain a biological sample (for example, the blood sample) from the subject before administration of the inhibitor. In some embodiments, the practitioner may obtain a biological sample (for example, a blood sample) from the subject after administration of the inhibitor to the subject. In some embodiments, the post-treatment sample can be obtained from the subject less than 48 (for example, less than 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 15 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight , seven, six, five, four, three, two or even less than one) hours after administration of the inhibitor to the subject. In some embodiments, the post-treatment sample can be obtained from the subject less than 20 (for example, less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four, three, two or one) days after administration to the subject of the inhibitor. In some embodiments, the biological sample is obtained from the subject no more than 20 (for example, no more than 19, 18, 20 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six , five, four, three, two or one) days after the inhibitor is administered to the subject.

In some embodiments, more than one practitioner can perform various stages of the methods described herein. For example, a practitioner can analyze (for example, measure the concentration or activity of one or more SUHa biomarker proteins in) the pre and post-treatment samples obtained from the subject. Another practitioner may receive information regarding the analysis of the samples by the first practitioner to determine in this way whether, for example, the subject has responded to treatment with a complement inhibitor. In some embodiments, another practitioner may obtain a pretreatment biological sample from a patient and a fourth practitioner may obtain a post-treatment biological sample from the subject. In some embodiments, all stages are carried out by the same practitioner.

Biological samples and sample collection

Biological samples suitable for use in the methods described herein include, for example, any biological liquid. A biological sample may be, for example, a test sample obtained from a subject (for example, a mammal such as a human being) or may be derived from said subject. A biological sample can also be a biological liquid such as urine, whole blood or a fraction thereof (for example, plasma or serum), saliva, semen, sputum, cerebrospinal fluid, tears or mucus. A biological sample can be further fractionated, if desired, to a fraction containing particular analytes (for example, 40 proteins) of interest. For example, a whole blood sample can be fractionated in serum or in fractions containing particular types of proteins. If desired, a biological sample may be a combination of different biological samples of a subject such as a combination of two different liquids.

The biological samples suitable for the invention may be new or frozen samples collected from a subject, or archival samples with diagnosis, treatment and / or history of known results. Biological samples can be obtained from a subject, for example, a subject who has, is suspected of having or is at risk of developing, a disorder associated with complement such as SUHa. Any suitable method can be employed to obtain the biological samples, although exemplary methods include, for example, phlebotomy, hyssop (eg, oral swab), washing or fine needle aspiration biopsy procedure. Biological samples can also be obtained from bone marrow.

In some embodiments, a protein extract can be prepared from a biological sample. In some embodiments, a protein extract contains the total protein content. Protein extraction methods are well known in the art. See, for example, Roe (2001) "Protein Purification Techniques: A Practical Approach", 55 2nd Edition, Oxford University Press. Many different and versatile kits can be used to extract proteins from body fluids and tissues, and are commercially available from, for example, BioRad Laboratories (Hercules, CA), BD Biosciences Clontech (Mountain View, CA), Chemicon International, Inc. (Temecula, CA), Calbiochem (San Diego, CA), Pierce Biotechnology (Rockford, IL) and Invitrogen Corp. (Carlsbad, CA).

 60

Those skilled in the art are well aware of methods for obtaining and / or storing samples that retain the activity or integrity of cells in the biological sample. For example, a biological sample may additionally contact one or more additional agents such as buffers and / or appropriate inhibitors, including protease inhibitors, agents that are intended to retain or minimize changes (e.g., changes in osmolarity or pH) in protein structure. Such inhibitors include, for example, chelants, such as ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), protease inhibitors such as

phenylmethylsulfonyl fluoride (PMSF), aprotinin and leupeptin. Buffers and conditions suitable for storing or otherwise manipulating whole cells are described, for example, in Pollard and Walker (1997), "Basic Cell Culture Protocols," volume 75 of Methods in molecular biology, Humana Press; Masters (2000) “Animal cell culture: a practical approach,” volume 232 of the Practical approach series, Oxford University Press; and Jones (1996) "Human cell culture protocols," volume 2 of Methods in molecular medicine, Humana Press. 5

A sample can also be processed to eliminate or minimize the presence of interfering substances. For example, a biological sample can be fractionated or purified to remove one or more materials (eg, cells) that are not of interest. Methods for fractionating or purifying a biological sample include, but are not limited to, flow cytometry, classification of fluorescence activated cells and sedimentation. 10

Complement inhibitors

Any compound that binds with and inhibits, or otherwise inhibits, the generation and / or activity of any of the human complement components according to the present disclosure can be used. For example, a complement inhibitor may be, for example, a small molecule, a nucleic acid or nucleic acid analog, a peptidomimetic or a macromolecule that is not a nucleic acid or a protein. These agents include, but are not limited to, small organic molecules, RNA aptamers, L RNA aptamers, Spiegelmers, antisense compounds, double stranded RNA, small interference RNA, blocked nucleic acid inhibitors and peptide nucleic acid inhibitors. In some embodiments, a complement inhibitor may be a protein or a protein fragment.

In some embodiments, the compositions contain antibodies specific to a human complement component. Some compounds include antibodies directed against complement components C1, C2, C3, C4, C5, C6, C7, C8, C9, Factor D, Factor B, Factor P, MBL, MASP-1, MASP-2, properdin or a Biologically active fragment of any of the foregoing, thereby preventing the generation of anaphylactic activity associated with C5a and / or preventing assembly of the C5b-9 membrane attack complex.

The compositions may also contain naturally occurring or soluble forms of complement inhibitor compounds such as CR1, LEX-CR1, MCP, DAF, CD59, H factor, cobra venom factor, FUT-175, complestatin and K76 COOH. Other compounds that can be used to bind with or otherwise block the generation and / or activity of any of the human complement components include, but are not limited to, proteins, protein fragments, peptides, small molecules, RNA aptamers including ARC187 (which Archemix Corporation, Cambridge, MA), L-RNA aptamers, spiegelmers, antisense compounds, serine protease inhibitors, molecules that can be used in RNA interference 35 (iRNA) such as double stranded RNA including interfering RNA are available on the market small (siRNA), blocked nucleic acid inhibitors (LNA), peptide nucleic acid (PNA) inhibitors, etc.

In some embodiments, the complement inhibitor inhibits complement activation. For example, the complement inhibitor can bind with and inhibit the activation activity of the C1 complement (for example, 40 C1q, C1r or C1s) or the complement inhibitor can bind with and inhibit (for example, inhibit cleavage of) C2, C3 or C4. In some embodiments, the inhibitor inhibits the formation or assembly of the C3 convertase and / or C5 convertase of the alternative and / or classical complement pathways. In some embodiments, the complement inhibitor inhibits the formation of the terminal complement, for example, formation of the C5b-9 membrane attack complex. For example, an antibody complement inhibitor may include an anti C5 antibody. Said anti-C5 antibodies can interact directly with C5 and / or C5b, to inhibit the formation of and / or the physiological function of C5b.

In some embodiments, the compositions described herein may contain an inhibitor of the C5 human complement component (for example, an antibody, or antigen-binding fragment thereof, that binds with a C5 human complement component protein or a biologically active fragment thereof such as C5a or C5b). As used herein, a "C5 complement component inhibitor" is any agent that inhibits: (i) the expression, or appropriate intracellular traffic or secretion by a cell, of a C5 complement component protein; (ii) the activity of the C5 C5a or C5b cleavage fragments (for example, the binding of C5a with its related cellular receptors or the binding of C5b with C6 and / or other 55 components of the terminal complement complex; see above) ; (iii) the cleavage of a human C5 protein to form C5a and C5b; (iv) the appropriate intracellular traffic, or secretion by a cell, of a protein component of complement C5; or (v) the stability of the C5 protein or the mRNA encoding the C5 protein. Inhibition of the expression of the C5 complement component protein includes: inhibition of transcription of a gene encoding a human C5 protein; increased degradation of an mRNA encoding a human C5 60 protein; inhibition of the translation of an mRNA encoding a human C5 protein; increased degradation of a human C5 protein; inhibition of the appropriate processing of a pre-pro human C5 protein; or inhibition of appropriate traffic or secretion by a cell of a human C5 protein. Methods for determining if a candidate agent is an inhibitor of the human complement component C5 are known in the art and are described herein. 65

An inhibitor of the C5 human complement component may be, for example, a small molecule, a polypeptide, a polypeptide analog, a nucleic acid or a nucleic acid analog.

It is understood that "small molecule" as used herein, refers to an agent, which has a molecular weight preferably of less than about 6 kDa and more preferably less than about 5 2.5 kDa. Many pharmaceutical companies have extensive libraries of chemical and / or biological mixtures that comprise matrices of small molecules, often fungal, bacterial or algae extracts, which can be explored with any of the assays in the application. This application contemplates using, among other things, small chemical libraries, peptide libraries or collections of natural products. Tan et al. have described a library with more than two million synthetic compounds that is compatible with tests based on miniaturized cells (J Am Chem Soc (1998) 120: 8565-8566). It is within the scope of the present application that said library can be used to scan for agents that bind with a target antigen of interest (eg, complement component C5). There are numerous libraries of compounds available in the market, such as the Chembridge DIVERSet. Also available are libraries of academic researchers, such as the Diversity suite of the NCI therapeutic development program. 15 Rational drug design can also be used. For example, the rational design of drugs can employ the use of crystalline structural information or solution on the component protein of the human complement C5. See, for example, the structures described in Hagemann et al. (2008) J Biol Chem 283 (12): 7763-75 and Zuiderweg et al. (1989) Biochemistry 28 (1): 172-85. The rational drug design can also be achieved based on known compounds, for example, a known C5 inhibitor (for example, an antibody, or antigen-binding fragment thereof, that binds with a C5 human complement component protein) .

Peptidomimetics may be compounds in which at least a part of an object polypeptide is modified, and the three-dimensional structure of the peptidomimetic remains substantially the same as that of the object polypeptide. The peptidomimetics may be analogs of a polypeptide object of the disclosure that are, in themselves, polypeptides containing one or more substitutions or other modifications within the subject polypeptide sequence. Alternatively, at least a part of the object polypeptide sequence can be replaced with a non-peptide structure, so that the three-dimensional structure of the object polypeptide is substantially conserved. In other words, one, two or three amino acid residues within the object polypeptide sequence can be replaced by a non-peptide structure. In addition, other peptide parts of the object polypeptide 30 can be replaced, but do not need to be replaced, with a non-peptide structure. Peptidomimetics (both peptide and non-peptide analogs) may have improved properties (eg, reduced proteolysis, increased retention or increased bioavailability). Peptidomimetics generally have improved oral availability, which makes them especially suitable for the treatment of disorders in a human or animal being. It should be noted that peptidomimetics may or may not have similar two-dimensional chemical structures, but they share common three-dimensional structural elements and geometry. Each peptidomimetic may also have one or more unique additional binding elements.

Nucleic acid inhibitors can be used to bind with and inhibit a target antigen of interest. The nucleic acid antagonist can be, for example, an aptamer. Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind to almost any molecule, including cell surface proteins. The systematic evolution of ligands by exponential enrichment process (SELEX) is potent and can be used to easily identify such aptamers. Aptamers can be prepared for a wide range of proteins important for therapy and diagnosis, such as growth factors and cell surface antigens. These oligonucleotides bind with their targets with similar affinities and specificities to the antibodies (see, for example, Ulrich (2006) Handb Exp Pharmacol. 173: 305-326).

In some embodiments, the complement inhibitor is a framework protein other than antibody. These proteins are obtained, in general, by adaptation based on combinatorial chemistry of preexisting antigen binding proteins. For example, the human transferrin binding site for human transferrin receptor 50 can be modified using combinatorial chemistry to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. Ali et al. (1999) J Biol Chem 274: 24066-24073. The part of human transferrin not involved in binding with the receptor remains unchanged and acts as a framework, as conserved framework regions of antibodies, to present the varying binding sites. Libraries are then scanned, like an antibody library, against a target antigen of interest to identify variants that have optimal selectivity and affinity for the target antigen. It is proposed that non-antibody scaffold proteins, although similar in function to antibodies, have several advantages compared to antibodies, including such advantages, among other things, enhanced solubility and tissue penetration, lower manufacturing cost and ease of conjugation with other molecules of interest. Hey et al. (2005) TRENDS Biotechnol 23 (10): 514-522. 60

One skilled in the art would appreciate that the framework portion of the non-antibody framework protein may include, for example, all or a portion of: the Z domain of S. aureus protein A, human transferrin, tenth fibronectin domain of human type III, kunitz domain of a human trypsin inhibitor, human CTLA-4, an ankyrine repeat protein, a human lipocalin, human crystalline, human ubiquitin or a trypsin inhibitor of E. elaterium. Same reference.

In some embodiments, the complement inhibitor is an antibody, or antigen-binding fragment thereof, that binds with a C5 human complement component protein. (Hereinafter, the antibody may sometimes be referred to as "anti C5 antibody").

In some embodiments, the anti-C5 antibody can bind with an epitope in the alpha chain of the 5-component human complement protein C5. Antibodies that bind with the C5 alpha chain are described, for example, in PCT Application Publication No. WO 2010/015608 and United States Patent No. 6,355,245. In some embodiments, the anti-C5 antibody can bind with an epitope in the beta chain of the C5 human complement component protein. Antibodies that bind with the C5 beta chain are described, for example, in Moongkarndi et al. (1982) Immunobiol 162: 397; Moongkarndi et al. (1983) Immunobiol 165: 323; and 10 Mollnes et al. (1988) Scand J Immunol 28: 307-312.

Additional exemplary antigenic fragments of the human complement component C5 are disclosed, for example, in US Patent No. 6,355,245.

 fifteen

Additional anti C5 antibodies, and antigen binding fragments thereof, suitable for use in the fusion proteins described herein, are described, for example, in PCT Application Publication No. WO 2010/015608.

In some embodiments, the anti-C5 antibody specifically binds to a component protein of the human C5 complement (for example, the human C5 protein having the amino acid sequence depicted in SEQ ID NO: 1). The terms "specific binding" or "specifically binding" refer to two molecules that form a complex (for example, a complex between an antibody and a protein component of complement C5) that is relatively stable under physiological conditions. Typically, binding is considered specific when the association constant (Ka) is greater than 106 M-1. Therefore, an antibody can specifically bind with a C5 protein with a Ka of at least (or more than) 106 (for example, at least or more than 107, 108, 109, 1010, 1011, 1012, 1013, 1014 or 1015 or greater) M-1. Examples of antibodies that specifically bind to a C5 human complement component protein are described in, for example, U.S. Patent No. 6,355,245.

The anti C5 antibodies described herein may have activity in the generation block or activity of the active fragments C5a and / or C5b of a component protein of complement C5 (eg, a human C5 protein). Through this blocking effect, anti-C5 antibodies inhibit, for example, the pro-inflammatory effects of C5a and the generation of the C5b-9 membrane attack complex (CAM) on the surface of a cell. Anti-C5 antibodies that have the ability to block the generation of C5a are described, for example, in Moongkarndi et al. (1982) Immunobiol 162: 397 and Moongkarndi et al. (1983) Immunobiol 165: 323. 35

In some embodiments, an anti-C5 antibody, or antigen-binding fragment thereof, can reduce the ability of a C5 protein to bind with the human complement component C3b (eg, C3b present in a C5 RA or RC convertase complex) ) in more than 50 (for example, more than 55, 60, 65, 70, 75, 80, 85, 90 or 95 or more)%. In some embodiments, upon binding with a C5 protein, the anti-C5 antibody or antigen-binding fragment thereof can reduce the ability of the C5 protein to bind with the complement component C4b (eg, C4b present in a convertase C5 RC) in more than 50 (for example, more than 55, 60, 65, 70, 75, 80, 85, 90 or 95 or more)%. Methods for determining if an antibody can block the generation or activity of active fragments C5a and / or C5b of a protein component of complement C5, or binding with component of complement C4b or C3b, are known in the art, and are described, for example. , in US Patent No. 6,355,245 and Wurzner et al. (1991) Complement Inflamm 8: 328-340.

In some embodiments, the composition comprises, and / or the antibody is, eculizumab (Soliris®, Alexion Pharmaceuticals, Inc., Cheshire, CT). (See, for example, Kaplan (2002) Curr Opin Investig Drags 3 (7): 1017-23; Hill (2005) Clin Adv Hematol Oncol 3 (11): 849-50; and Rother et al. (2007) Nature Biotechnology 25 (11): 1256-1488). In some embodiments, the composition comprises, and / or the antibody is, pexelizumab (Alexion Pharmaceuticals, Inc., Cheshire, CT). (See, for example, Whiss (2002) Curr Opin Investig Drugs 3 (6): 870-7; Patel et al. (2005) Drugs Today (Barc) 41 (3): 165-70; and Thomas et al. ( 1996) Mol Immunol 33 (17-18): 1389-401.)

In some embodiments, the C5 inhibitor is an antibody that binds with C5a (sometimes referred to herein as "anti C5a antibody"). In some embodiments, the antibody binds with C5a, but not with full-length C5. In some embodiments, the binding of an antibody with C5a can inhibit the biological activity of C5a. Methods for measuring C5a activity include, for example, chemotaxis, RIA or ELISA assays (see, for example, Ward and Zvaifler (1971) J Clin Invest 50 (3): 606-16 and Wurzner et al. (1991 ) Complement Inflamm 8: 328-340). In some embodiments, the binding of an antibody with C5a can inhibit the interaction between C5a and C5aR1. Suitable methods for detecting and / or measuring the interaction between C5a and C5aR1 (in the presence and absence of an antibody) are known in the art and are described, for example, in Mary and Boulay (1993) Eur J Haematol 51 (5): 282-287; Kaneko et al. (1995) Immunology 86 (1): 149-154; Giannini et al. (1995) J Biol Chem 270 (32): 19166-19172; and United States Patent Application Publication No. 20060160726. For example, the binding of detectably labeled C5a (eg radiolabeled) with peripheral blood mononuclear cells expressing C5aR1 can be evaluated in the presence and absence of an antibody A reduction

of the amount of detectably labeled C5a that binds with C5aR1 in the presence of the antibody, compared to the amount of binding in the absence of the antibody, is an indication that the antibody inhibits the interaction between C5a and C5aR1. In some embodiments, the binding of an antibody with C5a can inhibit the interaction between C5a and C5L2 (see below). Methods for detecting and / or measuring the interaction between C5a and C5L2 are known in the art and are described, for example, in Ward (2009) J Mol Med 87 (4): 375-378 and Chen et al. 5 (2007) Nature 446 (7132): 203-207 (see below).

In some embodiments, the C5 inhibitor is an antibody that binds with C5b (sometimes referred to herein as "an anti C5b antibody"). In some embodiments, the antibody binds with C5b, but does not bind with full-length C5. The structure of C5b is described, for example, in Müller-Eberhard (1985) 10 Biochem Soc Symp 50: 235-246; and Yamamoto and Gewurz (1978) J Immunol 120 (6): 2008-2015. As described above, C5b combines with C6, C7 and C8 to form the C5b-8 complex on the surface of the target cell. Intermediates of protein complexes formed during the series of combinations include C5b-6 (including C5b and C6), C5b-7 (including C5b, C6 and C7) and C5b-8 (including C5b, C6, C7 and C8). After the binding of several C9 molecules, the membrane attack complex (CAM, terminal complement complex (CCT) 15 C5b-9) is formed. When sufficient numbers of CAMs are inserted into target cell membranes, the openings they create (CAM pores) mediate in the rapid osmotic lysis of the target cells.

In some embodiments, the binding of an antibody with C5b can inhibit the interaction between C5b and C6. In some embodiments, binding of the antibody with C5b may inhibit the assembly or activity of the CAM-CCT C5b-9. In some embodiments, the binding of an antibody with C5b can inhibit complement-dependent cell lysis (eg, in vitro and / or in vivo). Suitable methods for evaluating whether an antibody inhibits complement-dependent lysis include, for example, hemolytic assays or other functional assays to detect the activity of soluble C5b-9. For example, a reduction in the ability of complement cell lysis in the presence of an antibody can be measured by a hemolysis assay described in Kabat and Mayer (eds.), Experimental 25 Immunochemistry, 2nd Edition, "135-240, Springfield, IL , CC Thomas (1961), pages 135-139, or a conventional variation of that assay such as the method of hemolysis of chicken erythrocytes as described, for example, in Hillmen et al. (2004) N Engl J Med 350 (6): 552.

Antibodies that bind with C5b as well as methods for preparing said antibodies are known in the art. 30 Commercially available anti-C5b antibodies are available from various suppliers including, for example, Hycult Biotechnology (catalog number: HM2080, clone 568) and Abcam ™ (ab46151 or ab46168).

Methods for determining if a particular agent is an inhibitor of the human complement component C5 are described herein and are known in the art. For example, the physiological concentration and / or activity of C5a and C5b in a biological liquid can be measured by methods well known in the art. Methods for measuring the concentration or activity of C5a include, for example, chemotaxis, RIA or ELISA assays (see, for example, Ward and Zvaifler (1971) J Clin Invest. 50 (3): 606-16 and Wurzner et al. . (1991) Complement Inflamm. 8: 328-340). For C5b, hemolytic assays or assays for soluble C5b-9 can be used as discussed herein. Other assays known in the art may also be used. Using assays of these suitable or other types, candidate agents capable of inhibiting the C5 human complement component such as an anti C5 antibody can be screened, for example, to identify compounds that are useful in the methods described herein and determine the appropriate dosage levels of said compounds.

 Four. Five

Methods for determining whether a candidate compound inhibits the cleavage of human C5 in C5a and C5b forms are known in the art and are described, for example, in Moongkarndi et al. (1982) Immunobiol 162: 397; Moongkarndi et al. (1983) Immunobiol 165: 323; Isenman et al. (1980) J Immunol 124 (1): 326-31; Thomas et al. (1996) Mol. Immunol 33 (17-18): 1389-401; and Evans et al. (1995) Mol. Immunol 32 (16): 1183-95.

 fifty

Inhibition of the human complement component C5 can also reduce the ability of complement cell lysis in the body fluids of a subject. Such reductions in the cell lysis capacity of the present complement can be measured by methods well known in the art, such as, for example, by a conventional hemolytic assay such as the hemolysis assay described in Kabat and Mayer (eds), "Experimental Immunochemistry , 2nd Edition, "135-240, Springfield, IL, Thomas Thomas (1961), pages 135-139, or a conventional variation of that assay such as the method of hemolysis of chicken erythrocytes as described, for example, in Hillmen et al. (2004) N Engl J Med 350 (6): 552.

Antibodies that bind with C3b and, for example, inhibit C3b convertase are also well known in the art. See for example, PCT application publications No. WO 2010/136311, WO 2009/056631 and WO 2008/154251. 60 Anti-C6 and anti-C7 antagonist antibodies have been described, for example, in Brauer et al. (1996) Transplantation 61 (4): 588-594 and U.S. Patent No. 5,679,345.

In some embodiments, the antibody is an anti factor B antibody (such as monoclonal antibody 1379 produced by ATCC Deposit No. PTA-6230). Anti-factor B antibodies are also described, for example, in 65 Ueda et al. (1987) J Immunol 138 (4): 1143-9; Tanhehco et al. (1999) Transplant Proc 31 (5): 2168-71; patents of

United States No. 7,999,082 and 7,964,705; and PCT Publication No. WO 09/029669.

In some embodiments, the antibody is an anti factor D antibody, for example, an antibody described in Pascual et al. (1990) J Immunol Methods 127: 263-269; Sahu et al. (1993) Mol Immunol 30 (7): 679-684; Pascual et al. (1993) Eur J Immunol 23: 1389-1392; Niemann et al. (1984) J Immunol 132 (2): 809-815; U.S. Patent No. 7,439,331; or U.S. Patent Application Publication No. 20080118506.

In some embodiments, the antibody is an antiproperdine antibody. Suitable antiproperdine antibodies are also well known in the art and include, for example, United States Patent Application Publications No. 20110014614 and PCT Application Publication No. WO2009110918. 10

Methods for treatment

Compositions for use in methods for treating or preventing SUHa in a subject (eg, a human being) are also provided herein. The compositions (for example, complement inhibitors and / or secondary agents) can be administered to a subject, for example, a human subject, using a variety of methods that depend, in part, on the route of administration. The route may be, for example, intravenous (IV) injection or infusion, subcutaneous (SC) injection, intraperitoneal (IP) injection or intramuscular injection.

Administration can be achieved by, for example, local infusion, injection or by means of an implant. The implant may be of a porous, non-porous or gelatinous material, including membranes, such as sialastic membranes or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, for example, United States Patent Publication No. 20080241223; U.S. Patent No. 5,501,856; 4,863,457; and 3,710,795; and European patents No. EP488401 and EP430539. The composition can be supplied to the subject by means of an implantable device based on, for example, diffusive, erodible or convective systems, for example, osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, steam pressure pumps, pumps electrolytics, effervescent pumps, piezoelectric pumps, erosion-based systems or electromechanical systems.

An adequate dose of a complement inhibitor (for example, an anti-C5 antibody or fragment thereof), whose dose can treat or prevent SUHa in a subject, may depend on a variety of factors including, for example, age, sex and the weight of a subject to be treated and the particular inhibitor compound used. For example, a different dose of a human C5 specific siRNA may be required to treat a subject with SUHa compared to the dose of an anti C5 antibody required to treat the same patient. Other factors that affect the dose administered to the subject include, for example, the type or severity of SUHa. For example, a subject having SUHa associated with CFH may require administration of a different dosage of the inhibitor than a subject with SUHa associated with MCP. Other factors may include, for example, other medical disorders that simultaneously affect or have previously affected the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, excretion rate, combination pharmacological and any other additional therapeutic product that is administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will depend on the criteria of the practically treating physician (for example, doctor or nurse).

The inhibitor can be administered as a fixed dose, or at a dose of milligram per kilogram "mg / kg". In some embodiments, the dose may also be chosen to reduce or prevent the production of antibodies or other immune responses of the host against one or more active agents in the composition. Although not intended to be limiting in any way, exemplary dosages of an inhibitor, such as an anti-C5 antibody, include, for example, 1-100 mg / kg, 0.5-50 mg / kg, 0.1- 100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg and 1-10 mg / kg body weight.

An anti-C5 antibody (eg, eculizumab) can be administered intravenously to a human being at a dose of about 900 mg every about 12 (e.g., about every 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42, or 49 or more) days. See, for example, Hill et al. (2005) Blood 106 (7): 2559.

An anti-C5 antibody (for example, eculizumab) can be administered intravenously at a dose of about 600 (e.g., about 625, 650, 700, 725, 750, 800, 825, 850, 875, 900, 55 925, 950 or 1,000 or more) mg each week, optionally, for two or more (for example, three, four, five, six, seven or eight or more) weeks. After the initial treatment, the antibody can be administered to the human being at a dose of about 900 mg every about 14 (for example, every about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 30, 42 or 49 or more) days, for example, as a maintenance dose. See, for example, Hillmen et al. (2004) N Engl J Med. 350 (6): 552-9 and Dmytrijuk et al. (2008) The Oncologist 13 (9): 993. 60

An anti-C5 antibody (eg eculizumab) can be administered intravenously to a human being at a dose of approximately 900 (for example, 925, 950, 975, 1000, 1100 or 1200 or more) mg each week, optionally, during two or more (for example, three, four, five, six, seven or eight or more) weeks. After initial treatment, the antibody can be administered to humans at a dose of approximately 1200 mg every 65 approximately 14 (for example, every 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 28, 30, 42 or 49

or more) days, for example, as a maintenance dose. See, for example, International Patent Application Publication No. WO 2010/054403.

As used herein, "administered chronically", "chronic treatment", "treat chronically" or similar grammatical variations thereof refer to a treatment regimen that is used to maintain a certain threshold concentration of a therapeutic agent in a patient's blood to completely or substantially suppress complement systemic activity in the patient for a prolonged period of time. Consequently, a patient chronically treated with a complement inhibitor can be treated for a period of time that is greater than or equal to 2 weeks (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 10 45, 46, 47, 48, 49, 50, 51 or 52 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months; or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7, 5, 8, 8.5, 9, 9.5, 10, 10.5 or 12 years or during the rest of the patient's life) with the inhibitor in an amount and with a dosage frequency that are sufficient to maintain a concentration of the inhibitor in the blood of the patient that substantially inhibits or inhibits the systemic activity of the complement in the patient. In some embodiments, the complement inhibitor may be administered chronically to a patient in need in an amount and with a frequency that is effective in maintaining serum hemolytic activity at less than or equal to 20 (e.g., 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5)%. See, for example, Hill et al. (2005) Blood 106 (7): 2559. The complement inhibitor can be administered to a patient in an amount and with a frequency that is effective in maintaining serum lactate dehydrogenase (LDH) levels within at least 20 (for example , 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5)% of the normal range for LDH. 20 See Hill et al. (2005) mentioned above. The complement inhibitor can be administered to the patient in an amount and with a frequency that are effective in maintaining a serum LDH level of less than 550 (e.g., less than 540, 530, 520, 510, 500, 490, 480, 470 , 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280 or less than 270) IU / l. To maintain systemic complement inhibition in a patient, the complement inhibitor can be administered chronically to the patient, for example, once 25 a week, once every two weeks, twice a week, once a day, once a month or once every three weeks.

A pharmaceutical composition may include a therapeutically effective amount of an inhibitor of the human complement component C5 (for example, an anti-C5 antibody or antigen-binding fragment thereof). Such effective amounts can be readily determined by a person skilled in the art based, in part, on the effect of the administered inhibitor, or the combinatorial effect of the antibody and one or more additional active agents, if more than one agent is used. A therapeutically effective amount of an inhibitor of the human complement component C5 (for example, an anti C5 antibody) can also vary according to factors such as the pathology, age, sex and weight of the individual, and the ability of the antibody (and one or more additional active agents) to induce a desired response in the individual, for example, relief of at least one condition parameter, for example, relief of at least one SUHa symptom. For example, a therapeutically effective amount of an inhibitor of the human complement component C5 (for example, an anti C5 antibody) can inhibit (reduce the severity of or eliminate the onset of) and / or prevent thrombocytopenia, microangiopathic hemolytic anemia, insufficiency renal and / or any of the SUHa symptoms known in the art or described herein. A therapeutically effective amount is also one in which any toxic or harmful effect of the composition is compensated by the therapeutically beneficial effects.

The terms "therapeutically effective amount" or "therapeutically effective dose" or similar expressions used herein are intended to mean an amount of an agent (eg, an inhibitor of the C5 human complement component) that induces the biological response or desired medical condition (for example, an improvement in one or more symptoms of SUHa). In some embodiments, a composition described herein contains a therapeutically effective amount of an inhibitor of the human complement component C5. In some embodiments, a composition described herein contains a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, that binds with a protein component C5 complement complement. In some embodiments, the composition contains two or more (for example, three, four, five, six, seven, eight, nine, 10 or 11 or more) different inhibitors of the human complement component C5 so that the composition as a whole Be therapeutically effective. For example, a composition may contain an antibody that binds with a human C5 protein and an siRNA that binds with, and promotes the degradation of an mRNA encoding a human C5 protein, in which the antibody and siRNA are each at 55 a concentration that when combined are therapeutically effective. In some embodiments, the composition contains the inhibitor and one or more second active agents so that the composition as a whole is therapeutically effective. For example, the composition may contain an antibody that binds with a human C5 protein and another agent useful for treating or preventing SUHa.

 60

The toxicity and therapeutic efficacy of said compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (animal models of SUHa). These procedures can be used, for example, to determine the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions, or inhibitors (eg, anti C5 antibodies) of the compositions, which show high therapeutic indices are preferred. Although they can

If compositions showing toxic side effects should be used, care should be taken to design a delivery system that directs said compounds to the site of affected tissue and minimizes potential damage to normal cells and, therefore, reduces side effects.

The data obtained from cell culture assays and animal studies can be used in the formulation of a dosage range for use in humans. Suitable animal models of SUHa are known in the art and described, for example, Atkinson et al. (2007) Journal of Experimental Medicine 204 (6): 1245-1248. The dosage of said inhibitors generally falls within a range of circulating concentrations of the inhibitors (for example, an anti-C5 antibody or antigen-binding fragment thereof) that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. For an inhibitor of the human complement component C5 (for example, an anti C5 antibody) used as described herein (for example, to treat or prevent SUHa), the therapeutically effective dose can be estimated initially from culture assays. mobile. A dose may be formulated in animal models to achieve a range of plasma concentration in circulation that includes the IC50 (ie, the concentration of the test compound that achieves a semi-maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

In some embodiments, the required dose of an inhibitor of the human complement component C5 can be determined based on the concentration of the human C5 protein in the subject's blood. For example, a subject that has a higher concentration of human C5 protein levels in circulation may require a higher dose of a human C5 inhibitor than a subject that has lower levels of human C5 in circulation. Methods for determining the concentration of the human complement component C5 in a sample of liquid derived from a subject's blood are known in the art and are described, for example, in Rawal et al. (1998) J Biol 25 Chem 273 (27): 16828-16835.

The methods can be performed together with other therapies for SUHa. For example, the composition can be administered to a subject at the same time as, before or after nephrectomy (for example, bilateral nephrectomy), dialysis, a plasma exchange or a plasma infusion (see, for example, Noris et al. . (2005) "Non-30 shiga toxin-associated hemolytic uremic syndrome." In: Zipfel P (ed). Complement and Kidney Disease. Basel: Birkhauser-Verlag, 65-83).

A "subject", as used herein, can be any mammal. For example, a subject can be a human being, a non-human primate (for example, monkey, baboon or chimpanzee), a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit , a guinea pig, a gerbil, a hamster, a rat or a mouse. In some embodiments, the subject is an infant (for example, a human infant).

As used herein, a subject "who needs prevention", "who needs treatment" or "who needs it" refers to one who, according to the criteria of the appropriate medical practitioner (for example, a doctor, a nurse) , or a nurse practitioner in the case of human beings; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as a treatment with a composition comprising an inhibitor of the human complement component C5) .

As used herein, a subject "who is at risk of developing SUHa" is a subject that has one or more (for example, two, three, four, five, six, seven or eight or more) factors of risk to develop the disorder. Risk factors for SUHa are well known in the art of medicine and include, for example, a predisposition to develop the condition, that is, a family history of the condition or a genetic predisposition to develop the condition such as, for example, one or more mutations in complement factor H (CFH), membrane cofactor protein (MCP; CD46), C4b binding protein, complement factor B (CFB) or complement factor I (CFI). See, for example, Warwicker et al. (1998) Kidney Int 53: 836-844; Richards et al. (2001) Am J Hum Genet 68: 485-490; Caprioli et al. (2001) Am Soc Nephrol 12: 297-307; Neuman et al. (2003) J Med Genet 40: 676-681; Richards et al. (2006) Proc Natl Acad Sci USA 100: 12966-12971; Fremeaux-Bacchi et al. (2005) J Am Soc Nephrol 17: 2017-2025; Esparza-Gordillo et al. (2005) Hum Mol Genet 14: 703-712; Goicoechea by Jorge et al. (2007) Proc Natl Acad Sci USA 104 (1): 240-245; Blom et al. (2008) J Immunol 180 (9): 6385-91; and Fremeaux-Bacchi 55 et al. (2004) J Medical Genet 41: e84). See also Kavanagh et al. (2006), mentioned above. Risk factors also include, for example, infection with Streptococcus pneumoniae, pregnancy, cancer, exposure to antineoplastic agents (e.g., quinine, mitomycin C, cisplatin or bleomycin), exposure to immunotherapeutic agents (e.g., cyclosporine, OKT3 or interferon ), exposure to antiplatelet agents (for example, ticlopidine or clopidogrel), HIV infection, transplantation, autoimmune disease and mixed methylmalonic aciduria and homocystinuria (cblC). See, for example, Constantinescu et al. (2004) Am J Kidney Dis 43: 976-982; George (2003) Curr Opin Hematol 10: 339-344; Gottschall et al. (1994) Am J Hematol 47: 283-289; Valavaara et al. (1985) Cancer 55: 47-50; Miralbell et al. (1996) J Clin Oncol 14: 579-585; Dragon-Durey et al. (2005) J Am Soc Nephrol 16: 555-63; and Becker et al. (2004) Clin Infect Dis 39: S267-S275. Therefore, a human being at risk of developing SUHa may be, for example, one who has a family history of SUHa and / or one who has an HIV infection. From the above it will be evident that the subjects “at risk of developing SUHa” do not

They are all subjects within a kind of interest.

A subject "suspected of having SUHa" is one who has one or more symptoms of the condition. Those skilled in the medical art are well aware of symptoms of this condition and these include, for example, severe hypertension, proteinuria, uremia, lethargy / fatigue, irritability, thrombocytopenia, microangiopathic hemolytic anemia and impaired renal function (for example, renal failure acute). It will be evident from the previous passage that the subjects "suspected of having HUS" are not all subjects within a species of interest.

SUHa can be genetic, acquired or idiopathic. SUHa can be considered genetic when two or more (for example, three, four, five or six or more) members of the same family are suffering from the disease at least 10 months apart and exposure to a triggering agent has been excluded common, or when one or more gene mutations associated with SUHa (for example, one or more mutations in CFH, MCP / CD46, CFB or CFI) are identified in a subject. For example, a subject may have SUHa associated with CFH, SUHa associated with CFB, SUHa associated with CFI or SUHa associated with MCP. Up to 30% of genetic SUHa is associated with mutations in CFH, 12% with mutations in MCP, 5-10% with mutations in CFI and less than 2% with 15 mutations in CFB. The genetic SUHa can be multiple (i.e., family; two or more affected family members) or simple (i.e., a single case in a family). SUHa can be considered acquired when an underlying environmental factor can be identified (for example, a drug, systemic disease, or viral or bacterial agents that do not result in Shiga-type exotoxin). SUHa can be considered idiopathic when no trigger (genetic or environmental) is evident. twenty

Methods may include identifying the subject as one who has, is suspected of having or is at risk of developing SUHa. In addition to the use of the SUHa biomarker profiles described herein, laboratory tests can be performed to determine if a human subject has thrombocytopenia, microangiopathic hemolytic anemia or acute renal failure. Thrombocytopenia can be diagnosed by a medical professional as one or more of: (i) a platelet count that is less than 150,000 / mm3 (for example, less than 60,000 / mm3); (ii) a reduction in the platelet survival time that is reduced, which reflects the enhanced platelet alteration in the circulation; and (iii) giant platelets observed in a peripheral smear, which is consistent with the secondary activation of thrombocytopoiesis. Microangiopathic hemolytic anemia can be diagnosed by a medical professional as one or more of: (i) hemoglobin concentrations that are less than 10 mg / dl (for example, less than 6.5 mg / dl); (ii) increased serum lactate dehydrogenase (LDH) concentrations (> 460 U / l); (iii) hyperbilirubinemia, reticulocytosis, free hemoglobin in circulation and low or undetectable haptoglobin concentrations; and (iv) the detection of fragmented red blood cells (schistocytes) with the typical appearance of spiny or hull-shaped cells in the peripheral smear along with a negative Coombs assay. See, for example, Kaplan et al. (1992) "Hemolytic Uremic Syndrome and Thrombotic 35 Thrombocytopenic Purpura," Informa Health Care (ISBN 0824786637) and Zipfel (2005) "Complement and Kidney Disease," Springer (ISBN 3764371668).

Blood concentrations of C3 and C4 can also be used as a measure of complement activation or deregulation. In addition, the condition of a subject can be further characterized by identifying that the subject harbors one or more mutations in a gene associated with SUHa such as CFI, CFB, CFH or MCP (mentioned above). Suitable methods for detecting a mutation in a gene include, for example, DNA sequencing and nucleic acid matrix techniques. See, for example, Breslin et al. (2006) Clin Am Soc Nephrol 1: 88-99 and Goicoechea de Jorge et al. (2007) Proc Natl Acad Sci USA 104: 240-245.

 Four. Five

The inhibitor of the human complement component C5 (for example, an anti-C5 antibody or antigen-binding fragment thereof) can be administered to a subject as a monotherapy. Alternatively, as described above, the inhibitor can be administered to a subject as a combination therapy with another treatment, for example, another treatment for SUHa. For example, combination therapy may include administering to the subject (for example, a human patient) one or more additional agents (for example, 50 antihypertensives) that provide a therapeutic benefit to the subject who has, or is at risk of developing, SUHa . The C5 human complement component inhibitor and the additional active agent (s) can be administered at the same time. Alternatively, the inhibitor may be administered first and the additional active agent (s) may be administered second. On the contrary, the additional active agent (s) can be administered first and the inhibitor can be administered second. 55

The C5 human complement component inhibitor may replace or augment a previously or simultaneously administered therapy. For example, after treating with an anti-C5 antibody or antigen-binding fragment thereof, administration of the additional active agent (s) can be stopped or reduced, for example, administered at lower levels. The administration of previous therapy can be maintained. A prior therapy 60 can be maintained until the level of human C5 inhibitor reaches a level sufficient to provide a therapeutic effect. The two therapies can be administered in combination.

The supervision of a subject (for example, a human patient) for an improvement in SUHa, as defined herein, means evaluating the subject with respect to a change in a disease parameter, for example, an improvement in one or more symptoms of the disease. Such symptoms include any of the symptoms.

of SUHa described herein. In some embodiments, the evaluation is performed at least 1 hour, for example, at least 2, 4, 6, 8, 12, 24 or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days , 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after starting treatment. The subject can be evaluated in one or more of the following periods; before the start of treatment; during the treatment; or after one or more elements of the treatment have been administered. The evaluation may include assessing the need for additional treatment, for example, assessing whether a dosage, frequency of administration or duration of treatment should be altered. It may also include assessing the need to add or reduce a selected therapeutic modality, for example, add or reduce any of the SUHa treatments described herein.

 10

Kits

The use of kits comprising various reagents and materials for carrying out the methods described herein is also provided. The procedures for measuring, diagnosing, evaluating and / or assessing described herein can be performed by diagnostic laboratories, experimental laboratories or individual practitioners. The invention provides the use of kits in any of or in all of these scenarios.

In some embodiments, the kits described herein comprise materials and reagents to, among other things, characterize or process biological samples (e.g., biological liquids), measure biomarker levels (e.g., levels of proteins or nucleic acids) , diagnose SUHa in a subject or monitor the response to treatment according to the methods provided in this document. In certain embodiments, a kit of the invention comprises at least one or more reagents that specifically detect protein levels of one or more SUHa biomarker proteins (eg, those selected from Table 1) and, optionally, instructions for using the kit. . The kit may include, for example, any of the matrices described herein. 25

In some embodiments, the kits may include suitable control samples (eg, biological fluids from normal healthy individuals or a solution comprising a known amount of control of a particular analyte of interest). In some embodiments, the kits of the invention may include instructions for using the kit according to one or more methods described herein and may comprise instructions for processing the biological sample (eg, a biological liquid) obtained from the subject and / or to perform the test or instructions to interpret the results.

The following examples are intended to illustrate, not limit, the invention.

 35

Examples

To better understand the pathology of SUHa, the inventors have collected samples of biological fluids (whole blood, serum, plasma and urine) from patients who have SUHa or are suspected of having SUHa both before and, at various points, after starting treatment with a complement inhibitor (anti-C5 antibody 40 eculizumab). An objective of this study was to define a series of clinically definable parameters that can be used to monitor the sensitivity of patients to treatment with the complement inhibitor as well as disease markers and progression or reduction thereof. The inventors have identified several proteins whose expression and / or activity was correlated with the pathology of SUHa and / or the sensitivity of a patient with SUHa to treatment with a complement inhibitor. The proteins were those involved or associated with complement and / or activation of endothelial cells, inflammation, kidney injury and coagulation (see Table 1).

For the study, a total of 41 adult subjects (27 women and 12 men) were recruited with a confirmed diagnosis of SUHa as well as normal healthy adult volunteers. All patients had SUHa confirmed on examination based on one or more of the following characteristics: platelet count less than 150 x 109 / l; 50 hemoglobin levels less than the lower limit of normal; LDH levels that were greater than or equal to 1.5 times the upper limit of normal; serum creatinine levels that were greater than or equal to the upper limit of normal; and an activity level of ADAMTS 13 that was greater than 5%. All patients had negative results for Shiga toxin assay.

 55

The average age of the patients at the time of inclusion was 40.3 years old. 68% of the patients were women; 2 (4.8%) were black or African American; and 1 patient (2.4%) was of Asian descent. Six patients (14.6%) reported a family history of SUHa. Twenty (48.7%) had at least one mutation of the complement regulatory protein identified or had a positive result for an autoantibody assay that binds with a complement regulatory protein. Thirty patients (73.2%) presented 60 a first clinical manifestation of SUHa. Six patients (14%) immediately started eculizumab without the use of plasma exchange / infusion (PE / PI). Twenty-four patients (58.5%) received baseline dialysis (before treatment with eculizumab). Nine patients (22%) had previously undergone a kidney transplant. Twenty-seven (66%) had a platelet count that was less than 150x109 / l. Thirty-two (78%) patients had a serum LDH level that was greater than the upper limit of normal. The mean levels of 65 haptoglobin (Hp) for the patients in this cohort was 0.6 ± 0.4 g / l; while creatinine levels in

Serum media in this cohort of patients was 411 ± 264.6 μmol / L (N = 40).

Biological fluids were collected at the time of admission to the study (before treatment) and then after treatment at each drug administration. Eculizumab was administered to the subjects in the following program: 900 mg once a week for four weeks; 1200 mg as the fifth dose; and 5 1200 mg once every two weeks thereafter for up to 55 weeks as part of a phase 2 clinical trial.

Example 1. Materials and methods

 10

Urine samples

Freshly collected urine was immediately mixed with protease inhibitors. Concentrations of various analytes including NGAL, cystatin C, clusterin, TIMP-1, β2 microglobulin, C5b9, C5a and urine creatinine collected from subjects were inhibited using commercially available kits as briefly described below.

NGAL levels were measured in urine using a commercially available kit (R&D Systems, Minneapolis, MN; catalog number: DLCN20). Briefly, urine samples were diluted 1: 3 using a calibrating diluent provided in the RD5-24 kit. 50 μl of each sample or conventional control of the kit (recombinant human lipocalin 2 20 expressed in NS0) was added to wells of a duplicate test plate, each well containing 100 μl of test diluent provided in the RD1-52 kit. After a two hour incubation at 4 ° C in the refrigerator, the wells were washed four times with 200 µl per well of wash solution. An enzyme-labeled anti-NGAL conjugate (horseradish peroxidase) was added at 200 µl per well and incubated for two hours at 4 ° C in the refrigerator. The wells were washed four times with 200 µl per well of wash solution and were revealed by adding 200 µl per well of TMB substrate solution provided in the kit (substrate for the anti-NGAL conjugate enzyme) and incubated at room temperature in darkness for 30 minutes. TMB is a rustican horseradish peroxidase substrate frequently used in ELISA. The reaction between the substrate and immobilized horseradish peroxidase (HRP) conjugated with antibodies in the ELISA wells produces a blue solution. After reaching the desired color intensity, the reaction is terminated by the addition of the termination solution (acidic), which changes the color of the solution from blue to yellow. Therefore, the reactions were stopped after incubation by adding 50 µl per well of termination solution provided in the kit to each well and the absorbance was read at 450 nm.

Cystatin C levels were measured with a commercially available kit (R&D Systems, Minneapolis, MN; catalog number 35: DSCTC0). Briefly, 1: 3 urine samples were diluted using calibrating diluent provided in the RD5-24 kit. 50 µl of each sample or conventional kit control (recombinant human CysC) was added to wells of a duplicate test plate, each well containing 100 µl of test diluent provided in the RD1-52 kit. After a two hour incubation at 4 ° C in the refrigerator, the wells were washed four times with 200 µl per well of wash solution. An enzyme labeled anti-CysC conjugate was added at 40 200 µl per well and incubated for two hours at 4 ° C in the refrigerator. The wells were washed four times with 200 µl per well of a wash solution and developed by adding 200 µl per well of TMB substrate solution provided in the kit (substrate for the enzyme of the anti-CysC conjugate) and incubated at room temperature in darkness for 30 minutes. The reactions were stopped after incubation by adding 50 µl per well of termination solution provided in the kit to each well and the absorbance at 450 nm was read. Four. Five

Clusterine levels were measured with a commercially available kit (R&D Systems, Minneapolis, MN; catalog number: DCLU00). Briefly, 1: 3 urine samples were diluted using calibrating diluent provided in the RD5T kit. 50 µl of each sample or conventional control of the kit (recombinant human clusterin) was added to wells of a duplicate test plate, each well containing 100 µl of test diluent 50 provided in the RD1-19 kit. After a two hour incubation at room temperature in the orbital shaker set at 500 rpm, the wells were washed four times with 200 µl per well of wash solution. An enzyme-labeled anti-clusterin conjugate at 200 µl per well was added and incubated for two hours at room temperature in the orbital shaker set at 500 rpm. The wells were washed four times with 200 µl per well of wash solution and developed by adding 200 µl per well of TMB substrate solution and incubated at room temperature in the dark for 30 minutes. The reactions were stopped after incubation by adding 50 µl per well of termination solution to each well and the absorbance at 450 nm was read.

TIMP-1 levels were measured with a commercially available kit (R&D Systems, Minneapolis, MN; catalog number: DTM100). Briefly, 1: 2 urine samples were diluted using calibrated diluent provided in the RD5P kit. 50 µl of each sample or conventional control of the kit (recombinant human TIMP-1) was added to wells of a duplicate test plate, each well containing 100 µl of test diluent provided in the RD1X kit. After a two hour incubation at room temperature in the orbital shaker set at 500 rpm, the wells were washed three times with 200 µl per well of wash solution. An enzyme-labeled anti-TIMP-1 conjugate was added at 200 µl per well and incubated for two hours at room temperature in the orbital shaker set at 500 rpm. The wells were washed four times with 200 μl per well of

Wash solution and were revealed by adding 200 µl per well of TMB substrate solution and incubated at room temperature in the dark for 30 minutes. The reactions were stopped after incubation by adding 50 µl per well of termination solution to each well and the absorbance at 450 nm was read.

Β2M levels were measured with a commercially available kit (R&D Systems, Minneapolis, MN; catalog number 5: DBM200). Briefly, urine samples were diluted 1:10 using sample diluent provided in the kit. 20 µl of each sample, kit controls or kit standards were added to duplicate wells, containing 100 µl of a solution containing enzyme-labeled anti-β2M conjugate. After one hour incubation at room temperature, the wells were washed six times with 200 µl per well of wash solution. The wells were revealed by adding 100 µl per well of TMB substrate solution and incubated at room temperature in the dark for 15 minutes. The reactions were stopped after incubation by adding 100 µl per well of termination solution to each well and the absorbance at 450 nm was read.

Creatinine levels were measured with a commercially available kit (R&D Systems, Minneapolis, MN; catalog number: KGE005). Briefly, 1:20 urine samples were diluted using water and 50 µl of 15 samples, kit controls or kit standards were added to duplicate wells, containing 100 µl of the alkaline picrate solution provided in the kit. After a 30 minute incubation at room temperature, the absorbance at 490 nm was measured.

C5b-9 levels were measured with a commercially available kit (BD Biosciences, San Jose, CA; catalog number 20: 558315) and a set of optEIA B reagent (BD Biosciences, San Jose, CA; catalog number : 550534). Briefly, an anti-C5b-9 1: 250 capture antibody was diluted in coating buffer, 100 µl of which was added to each well of a 96-well maxisorp plate (Nunc; catalog number: 439454) and incubated for one night at 4 ° C in the fridge. The wells were washed three times with 200 µl per well of wash solution and blocked by adding 200 µl per well of test diluent provided in the kit for 25 hours at room temperature. The wells were washed three times with 200 µl per well of wash solution and 100 µl of urine samples or kit standards were added to duplicates. After a two hour incubation at room temperature, the wells were washed three times with 200 µl per well of wash solution. 100 µl of the working solution of C5b-9 detector antibodies provided in the kit was added to each well and incubated for one hour at room temperature. The wells were washed seven times with 200 µl per well of wash solution and developed by adding 100 µl per well of TMB substrate solution and incubated at room temperature in the dark for 30 minutes. The reactions were stopped after incubation by adding 50 µl per well of a termination solution to each well and the absorbance at 450 nm was read.

C5a levels were measured with a commercially available kit (BD Biosciences, San Jose, CA; catalog number: 557965). Briefly, 100 µl of urine samples or kit standards were added to duplicate wells containing 50 µl of ELISA diluent provided in the kit. After a two hour incubation at room temperature, the wells were washed five times with 200 µl per well of wash solution. 100 µl of the working solution of C5a detecting antibodies provided in the kit was added to each well and incubated for one hour at room temperature. The wells were washed seven times with 200 µl per well of wash solution and developed by adding 100 µl per well of TMB substrate solution and incubated at room temperature in the dark for 30 minutes. The reactions were stopped after incubation by adding 50 µl per well of termination solution to each well and the absorbance at 450 nm was read.

Plasma 45

Plasma samples were prepared as follows. Blood was collected in a 10 ml BD ™ P100 tube (Becton Dickinson) containing EDTA. Whole blood was centrifuged no more than 60 minutes after collection in a refrigerated centrifuge (adjusted to maintain 4-8 ° C) for 10 minutes at 3,000 rpm. Plasma was then obtained from the sample after centrifugation. Hemolyzed samples were discarded. fifty

The concentration of various analytes including Ba, prothrombin fragment 1 + 2, thrombomodulin, vWF, sC5b-9 and C5a in blood plasma fractions collected from the subjects was measured using commercially available kits as briefly described below.

 55

Ba levels were measured with a commercially available kit (Quidel, San Diego, CA; catalog number: A033). Briefly, the wells of a test plate were washed three times with wash solution. 1: 1,000 plasma samples were diluted with test sample diluent provided in the kit and 100 µl of the diluted plasma samples, controls and kit standards were added to duplicate wells. After a 60 minute incubation at room temperature, the wells were washed five times with 200 µl per well of wash solution. 60 100 µl of an enzyme-labeled anti-Ba antibody conjugate was added to each well and incubated for sixty minutes at room temperature. After five washes with wash solution, 100 µl of TMB substrate was added to each well and incubated for fifteen minutes at room temperature protected from light. The reaction was stopped with the addition of 100 µl per well of termination solution and the absorbance at 450 nm was read.

 65

Prothrombin 1 + 2 fragment levels in EDTA plasma were measured with the Enzygnost F1 + 2 kit (Siemens

Healthcare; catalog number: OPBD03). Briefly, 1: 2 plasma samples were diluted with sample buffer and 50 µl of the diluted samples, or standard (containing a known concentration of recombinant human prothrombin 1 + 2 fragment) was added to the wells. After a 30 minute incubation at 37 ° C, the wells were washed three times with 200 µl per well of wash solution. 100 µl of an enzyme labeled prothrombin 1 + 2 fragment antibody conjugate was added to each well and incubated for 15 minutes at 37 ° C. After three additional washes, 100 µl of chromogenic substrate was added to each well and incubated for 15 minutes at room temperature protected from light. The reaction was stopped by adding 100 µl of termination solution to each well and the absorbance at 450 nm was read.

Plasma thrombomodulin (TM) levels were evaluated with EDTA with the TM ELISA kit (American 10 Diagnostics, Stamford, CT; catalog number: 837). Briefly, 1: 4 plasma samples were diluted with sample buffer and 200 µl of diluted or standard samples (containing a known concentration of recombinant TM) were added to the wells. After a 60 minute incubation at room temperature, the wells of the test plate were washed four times with 200 µl / well of wash solution. A solution of an enzyme labeled anti TM antibody (200 μl per well) was added and incubated for 30 minutes at room temperature. After 4 washes, 200 µl of substrate was added to each well and the wells were incubated for 20 minutes at room temperature protected from light. The reaction was stopped with 100 µl of 0.5 M H2SO4 and the absorbance at 450 nm was measured.

The levels of von Willebrand factor (vWF) activity in plasma with EDTA were determined by an ELISA kit using specific capture antibody for vWF collagen binding sites (American Diagnostica; catalog number: 885). Plasma samples and kit 1:20 controls were diluted with assay diluent and 100 µl of the diluted samples and controls were added to duplicate wells. After a 60 minute incubation at room temperature, the wells were washed 5 times with wash solution and 100 µl of an enzyme-labeled anti vWF conjugate was added to each well. The wells were incubated for 15 minutes at room temperature and washed 5 times with wash solution. 100 µl of TMB substrate (which, after cleavage by the enzyme, generates a detectable signal) was added to each well. The wells were incubated for 15 minutes at room temperature protected from light followed by the addition of 100 µl of termination solution provided in the kit to each well. The absorbance at 450 nm was measured in a period of 30 minutes from the addition of termination solution. 30

Circulation levels of sC5b-9 were determined with a human C5b-9 ELISA set (BD Biosciences, San Diego, CA; catalog number: 558315) and a set of optEIA BD B reagents (BD Biosciences; catalog number : 550534). Briefly, an anti-C5b-9 1: 250 capture antibody was diluted in coating buffer provided in the kit, 100 µl of which was added to wells of a 96-well maxisorp plate (Nunc) and incubated overnight at 4 ° C. After three washes in wash solution, the wells were blocked with 200 µl of test diluent provided in the kit for one hour at room temperature. After 3 more washes with wash solution, 100 µl of the plasma samples (diluted 1: 100 in test diluent) or standards (containing a known concentration of purified sC5b-9) were added and incubated for two hours at room temperature. The wells were washed three times with wash solution and 100 µl of 40 working detector was added (containing a biotin-labeled anti-C5b-9 detection antibody and streptavidin-labeled rustican peroxidase labeled 1: 250 in test diluent ) to each well. After one hour incubation at room temperature, the wells were washed seven times with wash solution and 100 µl of TMB substrate solutions were added to each well. The reaction was allowed to run for 30 minutes at room temperature protected from light. After the addition of 50 µl of termination solution to each well, the absorbance at 450 nm was determined.

Circulating levels of C5a were determined with a sandwich ELISA using the set of optEIA BD B reagents (BD Biosciences; catalog number: 550534). All incubations were performed in the presence of futane (BD Biosciences; catalog number: 550236). Briefly, an anti-C5a capture antibody was diluted to 50 2 μg / ml in coating buffer provided in the kit, 100 μl of which was added to wells of a 96-well maxisorp plate (Nunc) followed by overnight incubation. at 4 ° C. After three washes in wash solution, the wells were blocked with 200 µl of test diluent for one hour at room temperature. After 3 more washes with wash solution, 50 µl of plasma samples (diluted 1: 5 in test diluent) or standards (containing a known concentration of C5a) were added and incubated for 55 hours at room temperature. The wells were washed 4 times with wash solution and 100 µl of working detector was added by adding to each well (containing a biotin-labeled anti-C5a detection antibody and streptavidin-labeled rustican horseradish peroxidase diluted 1: 250 in diluent of test). After incubation for one hour at room temperature, the wells were washed seven times with wash solution and 100 µl of TMB substrate solutions were added to each well. The reaction was allowed to run for 30 minutes at room temperature protected from light. After the addition of 50 µl of termination solution to each well, the absorbance at 450 nm was measured.

Serum

 65

Serum samples were processed as follows. Blood was collected in a serum separator tube (SST)

10 ml vacutainer. The tube was inverted five times and the blood was allowed to clot at room temperature for at least 30 minutes, but not more than two hours. The tube was subjected to centrifugation at 1,800 rcf with the brake set. Hemolyzed samples were discarded.

The quantitative determination of various serum analytes was carried out using kits of Flex Matrix 5 sets of human cytometric beads (CBA) (Flex CBA set; Becton Dickinson Biosciences, San Diego, CA), and were acquired by flow cytometer ( FACS LSR II, Becton Dickinson) according to the manufacturer's instructions. A Flex set capture pearl is a unique population of pearls with defined fluorescence intensity and is coated with a specific capture antibody for a soluble protein. Each population of pearls is given an alphanumeric position designation, which indicates their position in relation to other pearls in the 10 Flex CBA BD system. Pearls with different positions in essays can be combined to create a multiple essay. In a Flex set test the capture bead, the PE conjugate detection reagent and the standard or test samples are incubated together to form sandwich-type complexes.

Briefly, standards were mixed for each analyte and serial dilution was performed using the assay diluent. Capture beads were prepared for each analyte and pooled using serum / plasma capture bead diluent. The serum samples were properly diluted and together with the standards were incubated with the mixed capture beads in a total volume of 100 µl for one hour at room temperature. Detecting phycoerythrin (PE) reagents for all analytes were mixed and added to the tubes (50 µl). The samples were washed with wash buffer after a two hour incubation at room temperature in the dark and were acquired by flow cytometer after reconstitution of the sediment in the wash buffer.

The following sets of beads were incubated with serum samples diluted 1: 4 in test diluent provided in the kit (in which the specified biomarker protein indicates the capture antibody conjugated with the bead): IFN-γ (Pearl E7; number catalog: 558283); IL-12 p70 (Pearl E5; catalog number: 558283); IL-25 1β (Pearl B4; catalog number: 558279); IL-6 (Pearl A7; catalog number: 558276); IL-8 (Pearl A9; catalog number: 558277); CXCL-9 (Pearl E8; catalog number: 558286); CXCL-10 (Pearl B5; catalog number: 558280); MCP-1 (Pearl D8; catalog number: 558287); VEGF (Pearl B8; catalog number: 558336); and sCD40L (Pearl C7; catalog number 560305). The following sets of beads were incubated with serum samples diluted 1:50 in diluent provided in the kit: ICAM-1 (Pearl A4; catalog number: 560269); VCAM-1 (Pearl 30 D6; catalog number: 560427); TNFR1 (Pearl C4; catalog number: 560156); E-selectin (Pearl D9; catalog number: 560419); P-selectin (Pearl D7; catalog number: 560426); and CCL5 (Pearl D4; catalog number: 558324).

Example 2: Results 35

Markers of continued complement activation

As summarized in Table 2 below, in relation to the concentration in a biological liquid sample of healthy volunteers, the plasma concentration of complement component Ba and sC5b9 and the urine concentration of C5a and sC5b-9 were elevated. in most patients with SUHa. See also Fig. 1.

Table 2.

 SUHa biomarker protein

 n / N (%) elevated at baseline P value

 Plasma ba

 35/35 (100.0) <0.0001

 SC5b-9 in plasma

 37/38 (97.4) <0.0001

 C5a in urine

 26/29 (89.7) 0.0007

 SC5b-9 in urine

 23/27 (85.2) 0.0025

 * "N" indicates the total number of patients evaluated for a given biomarker, and "n" indicates the number of "N" patients with elevated levels of the biomarker protein.

These results indicate that there is a significant activation of the complement of the systemic alternative route in patients with SUHa.

After treatment with eculizumab, the mean levels (concentrations) of these SUHa biomarkers were reduced (Figs. 1A-C). The mean levels of C5 and sC5b-9 in urine were significantly reduced to between 1 and 2.5 weeks after the start of treatment and remained so. The mean percentage reduction in the levels of 50 C5a in urine was greater than 40% at week 3 after treatment and more than 70% at week 6 (Fig. 1D). Urine sC5b-9 levels were reduced by more than 60% at week 3 (Fig. 1E). These markers normalized over time. Plasma Ba levels were also significantly reduced (p = 0.0053) to approximately four to six weeks after treatment with eculizumab, suggesting that over time eculizumab reduces the onset or amplification of the classical complement pathway (Fig. 1C). However, the 55 percent average reduction in plasma Ba levels was approximately 10% at week 6 and more than 30% at week 12 (Fig. 1F).

The percentage of patients with SUHa treated who experience normalized complement biomarker protein levels over time are shown in Figs. 2A-C. For example, as shown in Fig. 2B, 50% of treated SUHa patients show normalized levels of sC5b-9 in urine at 2.5 weeks after the start of treatment with eculizumab. At 17 weeks after the start of treatment, 79% of treated SUHa patients showed normalized sC5b-9 levels. However, BA levels are not normalized in most patients (Figs. 1C and 2C). These data indicate that even with eculizumab therapy there may be, in some patients, continued activation of the low-level complement.

Platelet activation and hemostatic markers

 10

As summarized in Table 3 below, in relation to the concentration in a biological liquid sample of healthy volunteers, the serum concentration of sCD40L and the plasma levels of prothrombin fragment 1 + 2 and D-dimer were significantly elevated in the majority of patients with SUHa. See also Figs. 3A-B.

 fifteen

Table 3.

 SUHa biomarker protein

 n / N (%) elevated at baseline P value

 sCD40L

 36/38 (94.7) <0.0001

 Prothrombin Fragment F1 + 2

 36/38 (94.7) <0.0001

 D-dimer

 34/36 (94.4) = 0.0002

 * "N" indicates the total number of patients evaluated for a given biomarker, and "n" indicates the number of "N" patients with elevated levels of the biomarker protein.

The release of sCD40L is generally associated with platelet metabolism and activity. Prothrombin F1 + 2 fragments are generated during the prothrombin to thrombin conversion, while the D-dimer is a fibrin degradation product that indicates fibrinolysis. twenty

After treatment with eculizumab, the mean levels (concentrations) of these SUHa biomarkers were reduced. The mean levels of plasma levels of F1 + 2 and D-dimer were significantly reduced to between 1 and 2.5 weeks (p = 0.0078 and 0.0083, respectively) after the start of treatment and remained so. As shown in Fig. 3C, the average reduction percentage in F1 + 2 was approximately 15% at week 3 25 and more than 50% at week 12. The average reduction percentage in D- levels dimer was approximately 40% at week 6, but greater than 60% at week 12. These data indicate that eculizumab therapy has an immediate effect on coagulation and fibrinolysis pathways. As shown in Figs. 4A-B, 32% of treated SUHa patients show normalization of F1 + 2 levels at week 26 after the start of treatment and 46% of patients have normalized levels of D-dimer. In contrast, levels 30 of sCD40L remained elevated throughout the study.

Markers of damage and / or activation of endothelial cells

As summarized in Table 4 below, in relation to the concentration in a sample of biological liquid from healthy volunteers, the plasma concentration of thrombomodulin and vWF and the serum concentration of VCAM-1 were significantly elevated in patients with SUHa. . See also Figs. 5A-C.

Table 4

 SUHa biomarker protein

 n / N (%) elevated at baseline P value

 Thrombomodulin

 33/34 (97.1) <0.0001

 VCAM-1

 36/38 (94.7) <0.0001

 Von Willebrand factor antigen

 15/38 (39.5) <0.02

 * "N" indicates the total number of patients evaluated for a given biomarker, and "n" indicates the number of those "N" patients with elevated levels of the biomarker protein, n.s. Indicates not significant.

 40

A high concentration of thrombomodulin and VCAM-1 in biological fluids of patients with SUHa indicates significant activation of endothelial cells. Thrombomodulin is released in response to C3a, which further underlines continued complement activation in patients with SUHa. The vWF concentration is also significantly high. vWF has several physiological roles including platelet adhesion and coagulation and is also a marker of damage and endothelial activation. Four. Five

After treatment with eculizumab, the mean levels (concentrations) of these SUHa biomarkers were reduced (Figs. 5A-C). The mean thrombomodulin and VCAM-1 levels were significantly reduced from baseline at week 17 (p = 0.0007 and <0.0001, respectively) after the start of treatment (see Figs. 6C and 6D). At week 26, the levels of VCAM-1 and vWF had also been reduced. However, although vWF was normalized in approximately 70% of patients with SUHa treated at week 17 after the start of treatment (Fig. 6B), thrombomodulin and VCAM-1 levels remained elevated. Interestingly, of the 10% of patients who normalized thrombomodulin levels (Fig. 6A), only one patient had

normalized thrombomodulin and vWF levels. These data indicate that eculizumab therapy has a rapid and robust positive effect to correct damage and activation of endothelial cells.

Inflammation markers

 5

Table 5 (below) shows a series of analytes detected in plasma and / or serum and indicates the percentage of patients with SUHa in which the respective analytes were elevated before treatment with complement inhibitor therapy.

Table 5 10

 SUHa biomarker protein

 n / N (%) elevated at baseline P value

 CXCL10 in serum

 23/38 (60.5) P <0.0001

 CXCL9 in serum

 33/38 (86.8) P <0.0001

 IL-18

 19/38 (50.0) P <0.0001

 MCP-1

 34/38 (89.5) P <0.0001

 TNFR1

 38/38 (100.0) P <0.0001

 VEGF

 25/38 (65.8) P <0.0001

 IL-6

 21/34 (61.8) P = 0.0019

 CCL5

 4/38 (10.5) P = 0.0045

 IFN-γ

 5/34 (14.7) P = 0.0353

 IL-8

 22/38 (57.9) n.s. (P = 0.0640)

 ICAM-1

 2/34 (5.9) n.s

 IL-1β

 1/38 (2.6) n.s

 IL-12p70

 2/34 (5.9) n.s

 * "N" indicates the total number of patients evaluated for a given biomarker, and "n" indicates the number of those "N" patients with elevated levels of the biomarker protein. ** The concentrations of the two analytes labeled "serum" were measured in serum. The concentrations of all other analytes in the table were measured in plasma. n.s. Indicates not significant.

Prior to eculizumab therapy, patients with SUHa had elevated levels of circulating inflammatory cytokines and chemokines including, for example, CXCL-10, CXCL-9, IL-18, TNFR1, MCP-1, VEGF, IL-6 e IL-8 After the start of treatment, however, TNFR1 was the earliest inflammatory marker to be significantly reduced (at week 6, p = 0.0012) (Fig. 7A). The mean concentration of TNFR1 remained significantly lower than the baseline in all subsequent visits (P <0.0001), but was only normalized in 6% of patients with SUHa (Fig. 7B). Similarly, the average levels of CXCL10 were significantly reduced at week 26 (p = 0.0055), but were not normalized in all patients with SUHa (31% of patients did not normalize). At week 26, the average IFN-γ levels normalized in approximately 50% of the patients; however, however, the average levels of IL-8 (p = 0.01), CXCL-9 20 (p = 0.01), IL-18 (p <0.0001) and VEGF (p <0, 0001) in serum they remained elevated in the majority of patients with SUHa, compared to normal controls, and were not significantly different from baseline. Serum IL-6 was significantly reduced (p = 0.04) from baseline at week 26 and remained elevated at week 26 compared to normal control.

 25

In contrast, the mean levels of CCL-5 rose significantly at week 17 after the start of treatment and then (p = 0.0072 and 0.0021 at weeks 12-17 and week 26, respectively). In response to vascular injury in mice, CCL5 is positively regulated, which promotes selective T-cell infiltration as part of a vascular wound healing response. See, for example, Rookmaaker et al. (2007) Am JPhysiol Renal Physiol 293 (2): F624-630. These data indicate that eculizumab therapy has a rapid and robust positive effect on the inflammation of many patients with SUHa, but that there may be low-level inflammation in these patients even during treatment.

Markers of tubular and glomerular kidney injury

 35

Table 6A (below) shows a series of analytes detected in urine collected from patients and indicates the percentage of patients with SUHa in which the respective analytes were elevated before treatment with complement inhibitor therapy.

Table 6A 40

 Biomarker

 n / N (%) elevated at baseline P value

 Beta-2 microglobulin (β2M)

 20/28 (71.4) P <0.0001

 Clusterin

 24/29 (82.8) P <0.0001

 Cystatin C

 18/29 (62.1) P = 0.0002

 TIMP-1

 22/29 (75.9) P = 0.0003

 FABP-1

 22/29 (75.9) P = 0.0130

 NGAL

 5/29 (17.2) P = 0.0151

 Biomarker

 n / N (%) elevated at baseline P value

 NAG

 3/23 (13.0) P = 0.0413

 CXCL10

 2/29 (6.9) n.s.

 CXCL9

 2/29 (6.9) n.s.

 KIM-1

 2/29 (6.9) n.s.

 * "N" indicates the total number of patients evaluated for a given biomarker, and "n" indicates the number of those "N" patients with elevated levels of the biomarker protein. n.s. Indicates not significant.

Prior to treatment with eculizumab, low molecular weight molecules that are normally filtered by the kidney were elevated in the urine of patients with SUHa including β2M, clusterin, cystatin C and NAG. The molecules produced by renal tubular epithelial cells in response to injury were also elevated, such as TIMP-1, NGAL and L-FABP. However, after treatment with eculizumab, CysC (p = 0.0012) (Fig. 8A), 5 clusterin (p = 0.0446) and TIMP-1 (p = 0.0353) are significantly reduced to 1-2 , 5 weeks after the start of treatment and remained significantly reduced throughout the course of the study. NGAL (p = 0.0003) (Fig. 8C), L-FABP (p = 0.0366) and NAG (p = 0.0369) were significantly reduced from baseline at 4-6 weeks after onset of treatment and remained so next. β2M was significantly reduced at 12-17 weeks (p = 0.0008) and thereafter (Fig. 8B). At week 26, the mean urine levels of all 10 analytes had normalized in the patients with SUHa treated.

These data indicate that eculizumab therapy has a rapid, robust and long-lasting positive effect that repairs tubular and glomerular kidney injury experienced by many patients with SUHa.

 fifteen

Summary

The following table provides a summary of exemplary SUHa biomarkers (although not an exhaustive list), which are elevated in patients with SUHa before treatment with eculizumab, but which are significantly reduced after treatment with eculizumab. The average time after the start of treatment with eculizumab in which there was a significant reduction of the SUHa biomarker was also provided in the table (Table 6B).

Table 6B

 Biomarker

 Week 1-2.5 Week 4-6 Week 12-17 Week 26

 U-C5a

 X

 U-C5b-9

 X

 F1 + 2

 X

 D-dimer

 X

 U-Cys-C

 X

 U-TIMP-1

 X

 Plasma ba

    X

 TNFR1

    X

 U-CLU

    X

 U-NGAL

    X

 Thrombomodulin

      X

 VCAM

       X

 L-FABP

       X

 B2M

       X

 CXCL10

          X

 IFN-γ

          X

 25

Baseline SUHa marker levels in SUHa patients receiving dialysis and / or receiving kidney transplant

Complement concentration, inflammation and markers of renal lesion in plasma and urine were also evaluated in patients with SUHa who received dialysis before eculizumab therapy. As shown in Table 7 (below 30), the mean concentration of TNFR1 in serum, Ba in plasma, C5b9, prothrombin fragments 1 + 2, β2M, clusterin, sC5b9, TIMP-1, NGAL, CysC and C5a was significantly elevated in patients with SUHa who underwent repeated dialysis (for example, two or more times in the 6 months prior to treatment) compared to patients with SUHa who did not experience repeated dialysis before inclusion in the study (before treatment ). See also Figs. 9A-E. 35

Table 7.

 Analyte

 Major with repeated dialysis

 TNFR1 (serum)

 p = <0.0001

 β2m (in urine)

 p = 0.0009

 Analyte

 Major with repeated dialysis

 Clusterin (in urine)

 p = 0.0020

 Ba (plasma)

 p = 0.0021

 C5b-9 (in urine)

 p = 0.0042

 TIMP-1 (in urine)

 p = 0.0070

 NGAL (in urine)

 p = 0.0110

 CysC (in urine)

 p = 0.020

 F1 + 2 (plasma)

 p = 0.0191

 C5b-9 (plasma)

 p = 0.0476

 C5a (in urine)

 p = 0.0477

 ** The concentration of the analyte labeled "serum" was measured in serum. The concentration of analytes designated with "in urine" were measured in urine, while the concentration of analytes labeled with "plasma" were measured in plasma obtained from the patients.

In addition, patients with SUHa who had received a kidney transplant before treatment with eculizumab had lower urine C5b-9 and baseline urine FABP-1 compared to patients who had not received a kidney transplant.

 5

Baseline SUHa marker levels vs. TMA markers

The levels of some biomarkers associated with SUHa in some patients with SUHa were correlated with abnormal thrombotic microangiopathy (TMA) markers such as reduced platelet counts, elevated LDH and increased haptoglobin levels. For example, patients with SUHa with reduced platelet counts 10 at baseline (<150,000 per μl of blood), showed elevated levels of cystatin C in urine (P = 0.0276) and clusterin in urine (P = 0.0401) . See Figs. 14A-B. SUHa patients who had elevated LDH levels showed increased levels of VCAM-1 (P = 0.0226) (Fig. 14C), d-dimer (P = 0.0369) (Fig. 14D), IL-18, thrombomodulin and TNFR1 (see below). High levels of haptoglobin were frequently present in patients with SUHa who had elevated levels of IL-18. fifteen

Baseline SUHa marker levels in SUHa patients receiving plasma therapy

Patients with SUHa with repeated plasma therapy before treatment with eculizumab showed higher mean levels of cystatin C in baseline urine (see Fig. 15). twenty

Correlations between biomarker levels and clinical parameters

Platelets

 25

A high level of CCL5 was positively correlated with higher platelet counts at baseline (p = <0.0001; cc (correlation coefficient) = 0.8106). An elevated sCD40L level was also correlated with higher platelet counts at baseline (p = <0.001; cc = 0.6313).

In addition, patients with normalized Ba levels after treatment with eculizumab show significantly higher platelet increases than patients whose Ba levels remain elevated after treatment. See Fig. 13.

Estimated glomerular filtration rate (eGFR), LDH and complement in urine

 35

A correlation was also observed between elevated plasma Ba levels and reduced eGFR (p <0.0001; cc = -0.7219). An elevated serum TNFR1 concentration of patients with SUHa before treatment was correlated with lactate dehydrogenase (LDH) levels (p = 0.027; cc = 0.3586), but was more significantly correlated with lower eGFR (p <0.0001 ; cc = -0.6134). In addition, the higher levels of complement components in urine C5a and sC5b-9 and renal injury markers (β2M, clusterin, cystatin C, NGAL and TIMP-1) were moderately correlated with lower eGFR (p = 0.0002 to 0 , 0242; cc = -0.4286 to -0.6714).

Elevated levels of sC5b-9, clusterin and TIMP-1 in urine were modestly correlated with proteinuria (p = 0.0086 to 0.0284; cc = 0.40 to 0.4788), while elevated levels of Ba in Plasma (p = 0.0017; cc = 0.517), β2M, clusterin, sC5b-9 in urine and cystatin C were correlated with increased creatinine in the urine of patients 45 before treatment with eculizumab (p = 0.0440-0, 0018; cc = 0.3882-0.6457).

First clinical presentation of SUHa

A correlation was also observed between patients experiencing their first manifestation of SUHa and 50 levels of urine D-dimer in plasma or FABP-1 in urine significantly elevated at baseline (before treatment with eculizumab) (see Table 8).

Table 8

 High biomarker (% n)

 Biomarker

 Individual SUHa manifestation Multiple p-value manifestations

 D-dimer in plasma (μg / l)

 27 (100.0) 7 (77.8) 0.0571

 FABP-1 in urine (ng / mg normalized with respect to creatinine)

 19 (90.5) 3 (37.5) 0.0079

Latent disease

 5

Six of the SUHa patients involved in the study presented at the time of inclusion normalized hematological parameters (including levels of haptoglobin, LDH and platelets). However, these patients still showed evidence of chronic inflammation and complement activation despite a stable clinical image. The patients had significantly elevated serum TNFR1 levels (as shown in Fig. 10A) as well as significantly elevated levels of thrombomodulin, Ba (Fig. 10B), prothrombin fragments 1 + 2 (Fig. 10 10E), VCAM- 1 (Fig. 10C) and d-dimer (Fig. 10D). Similarly, patients with normal platelet levels (> 150 x 109 platelets / μl) at baseline still show elevated levels of most biomarkers (for example, Ba (Fig. 10F), VCAM-1 (Fig. 10G ), D-dimer (Fig. 10H) and F1 + 2 (Fig. 10I)). Taken together, these findings indicate that, even for the subset of patients with SUHa that are considered to be in clinical remission after treatment, there are probably continued low levels of complement activity, coagulopathy 15 and inflammation.

Correlations between biomarker levels and clinical outcomes

Hematological responses 20

Patients with complete hematological responses show more drastic reductions in TNFR1, clusterin in urine and complement levels in urine (C5a and C5b-9) (Fig. 11). For example, 86% of patients who show a reduced concentration of these biomarker proteins of SUHa obtained a complete hematological response (normalization of platelets and LDH) in weeks 12-17 after the start of treatment with eculizumab. In addition, these patients showed a greater reduction in mean percentage of TNFR1 in serum, clusterin in urine, C5a in urine and C5b-9 in urine than patients who did not achieve a complete hematological response.

It was also observed that the rapidity of reduction of TNFR1 (for example, at week 12 compared to 30 week 17 or beyond) was correlated with a complete hematological response (p = 0.0008). The D-dimer normalization rate was significantly associated with a complete hematological response (p = 0.0109; cc = 6.26).

In addition, the data show that a significantly greater increase in platelet counts was achieved in weeks 12-17 (p = 0.0022) and week 26 (p = 0.0110) in patients with SUHa treated with eculizumab who have ( at weeks 12-17 and week 26, respectively) normalized plasma concentrations of Ba. Platelet improvement was also correlated with a significant reduction in average F1 + 2 levels at week 4-6 (P = 0.0148; cc = -0.4087) and week 12-17 (P = 0, 0073; cc = - 0.4396) and more modestly with a reduction in d-dimer levels in week 12-17 (P = 0.0470; cc = - 0.3381). However, a subset of 40 patients, despite demonstrating a greater increase in platelet counts in weeks four to 26, continued to show significantly elevated levels of prothrombin 1 + 2 fragments, thrombomodulin, β2M in urine, clusterin, TIMP-1 and cystatin C, suggesting continued underlying disease activity.

Analysis of the data collected from the study also revealed a correlation between the change in another concentration of biomarker protein and platelet recovery. For example, the concentration of CCL5, MCP-1 and sCD40L was positively correlated with increased platelet counts in patients treated with eculizumab as shown in Table 9 below.

Table 9. 50

 Week after the start of treatment with eculizumab

 Biomarker P-value Correlation coefficient

 1-2.5

 CCL-5 p <0.0001 0.7419

 sCD40L P = 0.0141 0.3950

 VEGF P = 0.0014 0.5002

 4-6

 CCL-5 p <0.0001 0.7743

 sCD40L p <0.0001 0.6818

 MCP-1 P = 0.0114 0.4169

 12-17

 CCL5 P = 0.0003 0.5656

 Week after the start of treatment with eculizumab

 Biomarker P-value Correlation coefficient

 26

 CCL-5 p <0.0001 0.7845

 sCD40L P = 0.0012 0.5398

Thrombomicroangiopathy (TMA)

Patients with SUHa treated with Eculizumab who have a greater reduction in plasma Ba levels more frequently achieved a complete TMA response (for example, the normalization of hematological parameters 5 (for example, platelet count and LDH levels) and conservation of renal function). For example, 72.7% of the patients obtained a complete TMA response at weeks 12-17 and 85.29% of the patients achieved a complete TMA response at week 26. As shown in Fig. 12, these patients showed a higher percentage of mean reduction in plasma Ba concentration than patients who did not obtain a complete TMA response (p = 0.0018 and p = 0.006, respectively). 10

eGFR after treatment

A relationship was also observed between the reduction and / or normalization of certain biomarkers and an improvement of eGFR. For example, a significantly greater improvement (Table 10) was observed in eGFR (for example, eGFR 15 ≥ 15 ml / min / 1.73 m2 maintained for at least two consecutive measurements obtained at least four weeks apart) among patients with MCP-1, IL-6 and IFN-γ normalized (at weeks 4-6); VCAM-1, CXCL10, CXCL9 and Ba normalized (at weeks 12-17) and Ba, β2M in urine, CysC in urine, vWF, D-dimer, clusterin, CXCL10, CXCL9, FABP-1 in urine and other standardized ( at week 26) (Table 10). See also Fig. 16. 20

Table 10

 Week after the start of treatment with eculizumab

 Standardized biomarker P value

 1-2.5

 * *

 4-6

 MCP-1 0.0002

 IL-6 0.0251

 VCAM-1 0.0166

 12-17

 VCAM-1 0.0003

 CXCL-10 0.0071

 Ba 0.0299

 CXCL-9 0.0441

 26

 VCAM-1 <0.0001

 Cystatin C <0.0001

 Ba 0.0002

 U-β2m 0.0013

 CXCL9 0.0027

 CXCL10 0.0172

 vWF 0.0052

 D-dimer 0.0224

 L-FABP 0.0230

 Clusterine 0.0300

 F1 + 2 0.0460

Example 3. Baseline levels of selected SUHa biomarker proteins in patients with SUHa 25

At baseline, prior to treatment with eculizumab, substantial evidence of significant complement activation, inflammation / vascular damage and organ injury was observed in patients with SUHa regardless of the use of plasma exchange / plasma infusion or normal laboratory values for counting platelet, Hp or LDH. As demonstrated by the data presented in Table 11, the concentrations of 30 SUHa biomarkers of complement activity, vascular inflammation, activation and endothelial damage, coagulation and renal injury were significantly elevated in patients with SUHa compared to healthy subjects.

Table 11. 35

 Disease process

 Biomarker (VSN interval; units) Median level in BL [interval] (P value vs. VSN **) n / N (%) elevated in BL Increase factor vs. VSN in BL

 CAP activation

 Plasma ba (388.0-588.0 ng / ml) 2676.4 [935.0 - 3668.01] (<0.0001) 35/35 (100) 5.53

 Disease process

 Biomarker (VSN interval; units) Median level in BL [interval] (P value vs. VSN **) n / N (%) elevated in BL Increase factor vs. VSN in BL

 Terminal complement activation

 U-C5a (0.0-0.7 ng / mg U-creat) 9.00 [0.3 - 76.6] (0.0007) 26/29 (89.7) 45

 U-sC5b-9 (0.0-0.6 ng / mg U-creat)

 30.50 [0.2 - 665.7] (0.0025) 23/27 (85.2) 305

 Inflammation

 sTNFRl (407.31-391.3 pg / ml) 17616.85 [4008.5 -54158.2] (<0.0001) 38/38 (100) 18.71

 Endothelial activation

 sVCAM-1 (159.2-444.7 ng / ml) 659.75 [375.4 - 1865.51 (<0.0001) 36/38 (94.7) 1.99

 Endothelial damage

 TM (2.0-3.6 ng / ml) 10 [3.4-24.1] (<0.0001) 33/34 (97.1) 3.64

 Coagulation

 F1 + 2 (82.9-305.5 pmol / l) 1017.55 [217.75774.01 (<0.0001) 36/38 (94.7) 5.46

 D-dimer (157.0-395.9 μg / l)

 2735 [330.0 - 44100.01 (0.0002) 34/36 (94.4) 9.84

 Kidney injury

 U-clusterin (5.7-437.1 ng / mg U-creat) 1232.30 [129.96091.2] (<0.0001) 24/29 (82.8) 8.62

 U-TIMP-1 (0.0-5.4 ng / mg U-creat)

 23.8 [1.4 -230.4] (0.0003) 22/29 (75.9) 39.67

 U-L-FABP-1 (0.0-16.9 ng / mg U-creat)

 58.00 [3.7-1309.8] (0.0130) 22/29 (75.9) 48.33

 U-β2m (0.0-2.7 μg / mg U-creat)

 18.4 [0.4-127.7] (<0.0001) 20/28 (71.4) 46

 U-cystatin-C (0.3-301.3 ng / mg U-creat)

 1256.9 [14.37189.61 (0.0001) 18/26 (69.2) 23.85

VSN means value or concentration of normal human being for a given SUHa biomarker protein indicated in the table.

"Creat" means creatinine, whose concentration is used to normalize certain concentrations of biomarkers indicated in the table. 5

CAP refers to the alternative path of the complement (see above).

"LB" refers to "baseline", that is, before treatment with eculizumab.

"N" is the total number of patients analyzed with respect to a given disease process and biomarker. "N" is the number of "N" patients in which a given biomarker was elevated.

"U" indicates that the analyte was measured in urine. 10

* P values were calculated using a Wilcoxon ranks sum test, testing with respect to a difference between groups.

In addition, the inventors have observed that there was no statistical significance between elevated baseline levels of certain SUHa biomarkers observed in patients who had received or were receiving plasma exchange therapy (PE) or plasma infusion (PI) in comparison with the level of increase of SUHa biomarkers in patients who did not receive PE or PI therapy. For example, the concentration of Ba, sTNFR1, sVCAM-1 and D-dimer was not reduced or normalized in patients who had received PE / PI therapy (Figs. 17A-D). Note that only 3 of 26 patients analyzed in the data presented in Figs. 17A-D did not receive PE / PI. Most patients (n = 23) had elevated levels of Cystatin C, compared with 20 normal healthy volunteers. Since Cystatin C is a marker of kidney injury (glomerular injury), it is possible that patients who did not receive PE / PI had less damage to their kidneys and therefore had reduced levels of biomarker proteins related to kidney damage in their urine.

Similarly, at baseline, before eculizumab therapy, the concentration of protein markers of complement activation (e.g., Ba), inflammation (e.g., sTNFR1), endothelial cell activation (sVCAM-1), coagulation (D-dimer) and renal injury (cystatin-C) was elevated in patients with SUHa who had normal platelet counts. See Figs. 18A-E. And patients with normal Hp and LDH levels showed evidence of complement activation, inflammation, endothelial cell activation, coagulation and continued renal injury (see Figs. 19A-E). 30

In view of the above, the concentration of biomarkers that reflect complement activity, vascular inflammation, activation and endothelial damage, coagulation and renal injury was chronically elevated in patients with SUHa compared to normal healthy subjects. Patients with SUHa who received PE / PI showed robust evidence of complement activation, vascular inflammation, endothelial activation, coagulation and significant continued renal injury. Although PE / PI may temporarily maintain normal platelet count and LDH in some patients, the previous results show that deregulation of the underlying complement and TMA processes persist. Despite normal laboratory values for platelet count, LDH and Hp, these studies indicate that there is complement activation, vascular inflammation, activation

Endothelial, coagulation and significant renal damage continued in patients with SUHa.

Example 4. Effects of sustained treatment with Eculizumab on SUHa biomarker concentrations

 5

The inventors have observed that treatment with sustained eculizumab inhibits the activation of chronic elevated complement and renal injury mediated by terminal complement, and reduces inflammation, endothelial damage and thrombotic risk in patients with SUHa. For example, treatment with sustained eculizumab rapidly and completely inhibited activation of the terminal complement as indicated by the reduction in the concentration of both C5a and sC5b-9 (for example, C5a and sC5b-9 in urine). See Figs. 20A-B. At baseline, patients with 10 SUHa showed significant terminal complement activation compared to VSN, despite the use of PE / PI or normal platelet counts in some patients. In fact, SUHa patients demonstrated 45 times higher urine C5a levels and 304 times higher urine sC5b-9 levels. However, during treatment with sustained eculizumab, all patients with SUHa demonstrated rapid and potent terminal complement blockade, with complete normalization of 15 terminal pathogen activation complement products and no difference in levels in relation to VSN.

In addition, treatment with sustained eculizumab normalized the concentration of biomarker proteins of renal lesion (Figs. 21A-C). Before starting therapy with eculizumab, most patients had elevated levels of biomarkers of: tubular interstitial lesion and impaired renal function (for example, L-FABP-1, ∼48 times greater than VSN), glomerular filtration ( for example, cystatin C, ∼24 times greater than VSN), proximal tubular lesion (for example, clusterin, 8.6 times greater than VSN). However, sustained treatment with eculizumab dramatically reduced urine concentrations of FABP-1 (up to 100%), cystatin C (up to 99%) and clusterin (up to 98%). This reduction was significant at all time points (P <0.0001 for all) and the reduced concentration of all markers of renal injury was not different from the levels in VSN. The 25 additional markers of renal injury (for example, TIM-1 and β2 microglobulin) were also normalized (see above in Example 2). These results suggest that ischemia and organ damage may be completely dependent on terminal complement and confirms clinical data demonstrating that sustained inhibition of complement-mediated TMA led to clinically significant eGFR improvement and dialysis arrest. 30

Sustained treatment with eculizumab also significantly reduces the activation of the alternative complement pathway (see Fig. 22). All patients with SUHa showed significant systemic CAP activation upstream of C5, with 5.5 times higher levels of Ba compared to VSN, before treatment with eculizumab. However, after the start of eculizumab therapy, the concentration of upstream biomarkers of CAP activation (eg, Ba levels) was reduced by 30% and the reduction after week 4-6 was significant in all the time points (p <0.005) compared to the concentration of the markers in VSN. Even so, Ba levels did not normalize in patients with SUHa treated with eculizumab, suggesting that CAP activation persists, reflecting deregulation of the underlying complement in patients with SUHa. To be clear, however, terminal complement blockade with eculizumab protected 40 patients from the clinical consequences of continued CAP activation.

In addition, chronic treatment of patients with SUHa with eculizumab resulted in significantly reduced concentrations of biomarkers associated with inflammation, endothelial activation and tissue damage (Figs. 23A-C). Serum sTNFR1 levels were elevated (18.7 times higher than VSN levels) in 100% of patients with baseline SUHa. Sustained treatment with eculizumab significantly reduced sTNFR1 to 94%. The reduction in the concentration of these biomarkers in week 4-6 was significant at all time points (P <0.0001). Soluble VCAM-1 and TM levels rose in> 95% of patients with SUHa at baseline 2 times and 3.6 times, respectively, compared to VSN, demonstrating significant endothelial cell activation and damage before eculizumab therapy. The 50 concentrations of TM and sVCAM-1 were also significantly reduced during treatment with eculizumab. After week 12-17, the reduction in the concentration of biomarkers of endothelial damage was significant at all subsequent time points (TM; P <0.0001), but still rose modestly compared to VSN. Dramatically reduced soluble TM levels may reflect the restoration of membrane bound TM, which protects against thrombotic risk. 55

Finally, chronic treatment with eculizumab rapidly and significantly reduced the concentration of biomarkers associated with thrombotic risk and coagulation (Figs. 24A-B). The concentration of coagulation biomarkers F1 + 2 and D-dimer was significantly elevated (5.5 times and 9.8 times higher than VSN) at baseline in more than 94% of patients with SUHa (P <0.0001 and P = 0.0002, respectively). Even so F1 + 2 and D-60 dimer were significantly reduced at 2.5 weeks after the start of treatment with eculizumab. The concentration of F1 + 2 was reduced by up to 88% (P <0.05 for all time points) and the concentration of D-dimer was reduced to 99% (P <0.0001 for all time points) with treatment with sustained eculizumab. However, these two markers remained modestly elevated above the respective concentrations in normal healthy subjects. 65

Claims (16)

1. A complement inhibitor, which is an anti-C5 antibody or a C5 binding fragment thereof, for use in a method for treating atypical hemolytic uremic syndrome (SUHa), the method comprising:  5  (a) determining the concentration of at least two biomarker proteins associated with SUHa in a biological liquid obtained from a subject that has, is suspected of having or is at risk of developing SUHa, in which the biomarker proteins associated with SUHa are TNFR1 and at least one other biomarker protein associated with SUHa selected from: CXCL10, MCP-1, IFN-γ, IL-6, a proteolytic fragment of the complement component component B, soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, d- dimer, thrombomodulin, VCAM-1, 10 von Willebrand factor (vWF), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP -1), albumin, CXCL9, KIM-1, soluble CD40 ligand (sCD40L), ICAM-1, IL-1 beta, IL-12 p70, IL-8 and vascular endothelial cell growth factor (VEGF); Y  (b) administering to the subject the complement inhibitor in an amount and with a frequency sufficient to reduce the concentration of the at least two biomarker proteins associated with SUHa, as compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with complement inhibitor. 2. The complement inhibitor for use according to claim 1, wherein the subject:  (a) has received dialysis at least once in the three months immediately prior to treatment with the complement inhibitor; or  (b) is one who experiences the first manifestation of acute SUHa.  25 3. The complement inhibitor for use according to claim 1 or 2, wherein the reduced concentration of the at least two biomarker proteins associated with SUHa occurs within two weeks or two months after the first administration of the inhibitor of the complement. 4. A method for monitoring the sensitivity of a subject to treatment with a complement inhibitor, which is an anti-C5 antibody or a C5 binding fragment thereof, the method comprising: determining the concentration of at least two biomarker proteins associated with SUHa in a biological liquid obtained from the subject, in which:  (a) the biomarker proteins associated with SUHa are TNFR1 and at least one other biomarker protein associated with SUHa selected from: CXCL10, MCP-1, IFN-γ, IL-6, a proteolytic fragment of the component component of complement B, soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, D-dimer, thrombomodulin, VCAM-1, von Willebrand factor (vWF), complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1 , NGAL, fatty acid binding protein 1 (FABP-1), albumin, CXCL9 and KIM-1; 40  (b) the subject has, is suspected of having or is at risk of developing SUHa;  (c) the subject is treated or is being treated with the complement inhibitor; Y  (d) a reduced concentration of the at least two biomarker proteins associated with SUHa compared to the concentration in a sample of biological liquid of the same type obtained from the subject before treatment with the complement inhibitor indicates that the subject is sensitive to treatment with the inhibitor Four. Five 5. The complement inhibitor for use according to any one of claims 1 to 3 or the method according to claim 4, wherein the subject is being chronically treated with a complement inhibitor.  fifty 6. The complement inhibitor for use according to any one of claims 1 to 3 or the method according to claim 4 or 5, wherein the antibody or antigen-binding fragment thereof:  (i) is selected from a humanized antibody, a recombinant antibody, a diabody, a chimerized or chimeric antibody, a monoclonal antibody, a deimmunized antibody, a completely human antibody, a single chain antibody, an Fv fragment, an Fd fragment, a Fab fragment, a Fab 'fragment and an F (ab') 2 fragment;  (ii) inhibits the cleavage of C5 in fragments C5a and C5b; or  (iii) is the eculizumab antibody or the pexelizumab antigen binding fragment.  60 7. A method for diagnosing a subject who has or is at risk of developing atypical hemolytic uremic syndrome (SUHa), the method comprising: determining the concentration of at least two biomarker proteins associated with SUHa in a biological liquid obtained from a subject, wherein the biomarker proteins associated with SUHa are TNFR1 and at least one other biomarker protein associated with SUHa selected from: a proteolytic fragment of the complement component component B, soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, 65 von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, microglobulin β2 (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, binding protein fatty acids 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor (VEGF), IL-6, albumin, IL-8 and CCL5 wherein a high concentration of the at least two biomarker proteins associated with SUHa compared to the concentration of biomarker proteins associated with SUHa in a control sample of biological liquid of the same type indicates that the subject has, or is at risk to develop, SUHa. 8. The complement inhibitor for use according to any one of claims 1 to 3 and 6 or the method according to any one of claims 4 to 7, wherein the at least one other biomarker protein associated with SUHa is selected of: a proteolytic fragment of factor B, C5a, soluble C5b-9 10 (sC5b-9), VCAM-1, thrombomodulin, prothrombin fragments 1 and 2 (F1 + 2), D-dimer, clusterin, TIMP-1, FABP-1, beta-2 microglobulin (b2m) and cystatin-C, in which VCAM-1 is soluble VCAM-1 (sVCAM-1). 9. The complement inhibitor for use according to any one of claims 1 to 3, 6 and 8 or the method according to any one of claims 4 to 8, wherein the at least two biomarker proteins associated with SUHa are measured using an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). 10. The complement inhibitor for use according to any one of claims 1 to 3, 6, 8 and 9 or the method according to any one of claims 4 to 9, wherein the biological liquid is blood, a 20 blood fraction, such as serum or plasma, or urine. 11. The complement inhibitor for use according to claim 10 or the method according to claim 10, wherein the blood fraction is serum.  25 12. The complement inhibitor for use according to any one of claims 1 to 3, 6 and 8 to 11 or a method according to any one of claims 4 to 11, wherein:  (i) the concentration of at least one biomarker associated with SUHa in two or more types of biological fluid is measured; or 30  (ii) the concentration of a first of the at least two biomarker proteins associated with SUHa in one type of biological liquid is measured and the concentration of a second of the at least two biomarker proteins of SUHa is measured in a second type of liquid. 13. The complement inhibitor for use according to any one of claims 1 to 3, 6 and 8 to 12 or 35 a method according to any one of claims 4 to 12, wherein:  (i) the concentration of CXCL9, CXCL10, IFN-γ, MCP-1, CCL5, sCD40L or TNFR1 in the subject's serum, wherein TNFR1 is soluble TNFR1 (sTNFR1) is determined;  (ii) the concentration of at least one of β2 (β2M) microglobulin, clusterin, cystatin C, NAG, 40 TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL10, CXCL9, albumin is determined and KIM-1 in the subject's urine;  (iii) the concentration of NGAL, a proteolytic fragment of complement component component B, soluble C5b9 (sC5b9), prothrombin fragment F1 + 2, D-dimer, thrombomodulin or von Willebrand Factor (vWF) in the plasma of the plasma is determined subject; and / or 45  (iv) the concentration of Ba in plasma obtained from the subject is determined. 14. The complement inhibitor for use according to any one of claims 1 to 3, 6 and 8 to 13 or a method according to any one of claims 4 to 13, wherein the TNFR1 is sTNFR1 and the concentration Normal control of sTNFR1 is less than 2000 pg / ml. fifty 15. The complement inhibitor for use according to any one of claims 1 to 3, 6 and 8 to 14 or the method according to any one of claims 4 to 14, wherein the TNFR1 is sTNFR1 and the concentration of sTNFR1 in the biological sample is considered high when it is:  55  (i) at least twice as high as the control concentration of sTNFR1; or  (ii) at least 10,000 pg / ml. 16. Use of a diagnostic kit in the diagnosis of SUHa, in which the diagnostic kit comprises: (a) a test plate and (b) at least three binding agents, in which each binding agent binds with a different biological analyte protein 60, in which the analyte proteins are selected from: a proteolytic fragment of the complement component component B, soluble C5b9 (sC5b9), thrombomodulin, VCAM-1, von Willebrand factor (vWF), soluble CD40 ligand (sCD40L), prothrombin fragment F1 + 2, D-dimer, CXCL10, MCP-1, TNFR1, IFN-γ, ICAM-1, IL-1 beta, IL-12 p70, complement component C5a, β2 microglobulin (β2M), clusterin, cystatin C, NAG, TIMP-1, NGAL, fatty acid binding protein 1 (FABP-1), CXCL9, KIM-1, IL-18, vascular endothelial cell growth factor 65 ( VEGF), IL-6, albumin, IL-8 and CCL5, and one of the proteins is TNFR-1. 17. The use according to claim 16, wherein the binding agent is an antibody or an antigen binding fragment thereof.